Wnt Antagonists and Methods of Treatment and Screening

ABSTRACT

The present invention relates to compositions comprising Wnt antagonists and methods of treating Wnt-associated diseases and disorders, such as cancer, inducing differentiation, and reducing the frequency of cancer stem cells, as well as novel methods of screening for such Wnt antagonists. In particular, the invention discloses soluble FZD, SFRP and Ror receptors and their use.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. ProvisionalApplication No. 61/294,270, filed Jan. 12, 2010, U.S. ProvisionalApplication No. 61/393,675, filed Oct. 15, 2010, and U.S. ProvisionalApplication No. 61/424,408, filed Dec. 17, 2010, each of which is herebyincorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:sequencelisting_ascii.txt, Size: 126 kilobytes; and Date of Creation:Aug. 25, 2011) is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides novel compositions and methods fortreating cancer and other Wnt-associated diseases or disorders, as wellas novel screening methods for identifying additional novel therapeuticagents. In particular, the present invention provides Wnt antagonistsincluding soluble receptor proteins useful for the treatment of solidtumors and other Wnt-associated diseases and conditions.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death in the developed world,with over one million people diagnosed with cancer and 500,000 deathsper year in the United States alone. Overall it is estimated that morethan 1 in 3 people will develop some form of cancer during theirlifetime. There are more than 200 different types of cancer, four ofwhich - breast, lung, colorectal, and prostate - account for over halfof all new cases (Jemal et al., 2009, Cancer J. Clin., 58:225-249).

The Wnt signaling pathway has been identified as a potential target forcancer therapy. The Wnt signaling pathway is one of several criticalregulators of embryonic pattern formation, post-embryonic tissuemaintenance, and stem cell biology. More specifically, Wnt signalingplays an important role in the generation of cell polarity and cell fatespecification including self-renewal by stem cell populations.Unregulated activation of the Wnt pathway is associated with numeroushuman cancers where it can alter the developmental fate of tumor cellsto maintain them in an undifferentiated and proliferative state. Thuscarcinogenesis can proceed by usurping homeostatic mechanismscontrolling normal development and tissue repair by stem cells (reviewedin Reya & Clevers, 2005, Nature, 434:843-50; Beachy et al., 2004,Nature, 432:324-31).

The Wnt signaling pathway was first elucidated in the Drosophiladevelopmental mutant wingless (wg) and from the murine proto-oncogeneint-1, now Wnt1 (Nusse & Varmus, 1982, Cell, 31:99-109; Van Ooyen &Nusse, 1984, Cell, 39:233-40; Cabrera et al., 1987, Cell, 50:659-63;Rijsewijk et al., 1987, Cell, 50:649-57). Wnt genes encode secretedlipid-modified glycoproteins of which 19 have been identified inmammals. These secreted ligands activate a receptor complex consistingof a Frizzled (FZD) receptor family member and low-density lipoprotein(LDL) receptor-related protein 5 or 6 (LRP5I6). The FZD receptors areseven transmembrane domain proteins of the G-protein coupled receptor(GPCR) superfamily and contain a large extracellular N-terminal ligandbinding domain with 10 conserved cysteines, known as a cysteine-richdomain (CRD) or Fri domain. There are ten human FZD receptors, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9 and FZD10. Different FZDCRDs have different binding affinities for specific Wnts (Wu & Nusse,2002, J. Biol. Chem., 277:41762-9), and FZD receptors have been groupedinto those that activate the canonical β-catenin pathway and those thatactivate non-canonical pathways described below (Miller et al., 1999,Oncogene, 18:7860-72). To form the receptor complex that binds the FZDligands, FZD receptors interact with LRP5/6, single pass transmembraneproteins with four extracellular EGF-like domains separated by six YWTDamino acid repeats (Johnson et al., 2004, J. Bone Mineral Res.,19:1749).

The canonical Wnt signaling pathway activated upon receptor binding ismediated by the cytoplasmic protein Dishevelled (Dsh) interactingdirectly with the FZD receptor and results in the cytoplasmicstabilization and accumulation of β-catenin. In the absence of a Wntsignal, β-catenin is localized to a cytoplasmic destruction complex thatincludes the tumor suppressor proteins adenomatous polyposis coli (APC)and Axin. These proteins function as critical scaffolds to allowglycogen synthase kinase-3β (GSK3β) to bind and phosphorylate β-catenin,marking it for degradation via the ubiquitin/proteasome pathway.Activation of Dsh results in phophorylation of GSK3β and thedissociation of the destruction complex. Accumulated cytoplasmicβ-catenin is then transported into the nucleus where it interacts withthe DNA-binding proteins of the TCF/LEF family to activatetranscription.

In addition to the canonical signaling pathway, Wnt ligands alsoactivate β-catenin-independent pathways (Veeman et al., 2003, Dev. Cell,5:367-77). Non-canonical Wnt signaling has been implicated in numerousprocesses but most convincingly in gastrulation movements via amechanism similar to the Drosophila planar cell polarity (PCP) pathway.Other potential mechanisms of non-canonical Wnt signaling includecalcium flux, JNK, and both small and heterotrimeric G-proteins.Antagonism is often observed between the canonical and non-canonicalpathways, and some evidence indicates that non-canonical signaling cansuppress cancer formation (Olson & Gibo, 1998, Exp. Cell Res., 241:134;Topol et al., 2003, J. Cell Biol., 162:899-908). Thus in certaincontexts, FZD receptors act as negative regulators of the canonical Wntsignaling pathway. For example, FZD6 represses Wnt3a-induced canonicalsignaling when co-expressed with FZD1 via the TAK1-NLK pathway (Golan etal., 2004, JBC, 279:14879-88). Similarly, FZD2 was shown to antagonizecanonical Wnt signaling in the presence of Wnt5a via the TAK1-NLK MAPKcascade (Ishitani et al., 2003, Mol. Cell. Biol., 23:131-39).

The canonical Wnt signaling pathway also plays a central role in themaintenance of stem cell populations in the small intestine and colon,and the inappropriate activation of this pathway plays a prominent rolein colorectal cancers (Reya & Clevers, 2005, Nature, 434:843). Theabsorptive epithelium of the intestines is arranged into villi andcrypts. Stem cells reside in the crypts and slowly divide to producerapidly proliferating cells that give rise to all the differentiatedcell populations that move out of the crypts to occupy the intestinalvilli. The Wnt signaling cascade plays a dominant role in controllingcell fates along the crypt-villi axis and is essential for themaintenance of the stem cell population. Disruption of Wnt signalingeither by genetic loss of TCF7/2 by homologous recombination (Korinek etal., 1998, Nat. Genet., 19:379) or overexpression of Dickkopf-1 (Dkk1),a potent secreted Wnt antagonist (Pinto et al., 2003, Genes Dev.,17:1709-13; Kuhnert et al., 2004, PNAS, 101:266-71), results indepletion of intestinal stem cell populations.

A role for Wnt signaling in cancer was first uncovered with theidentification of Wntl (originally inti) as an oncogene in mammarytumors transformed by the nearby insertion of a murine virus (Nusse &Varmus, 1982, Cell, 31:99-109). Additional evidence for the role of Wntsignaling in breast cancer has since accumulated. For instance,transgenic overexpression of β-catenin in the mammary glands results inhyperplasias and adenocarcinomas (Imbert et al., 2001, J. Cell Biol.,153:555-68; Michaelson & Leder, 2001, Oncogene, 20:5093-9) whereas lossof Wnt signaling disrupts normal mammary gland development (Tepera etal., 2003, J. Cell Sci., 116:1137-49; Hatsell et al., 2003, J. MammaryGland Biol. Neoplasia, 8:145-58). More recently mammary stem cells havebeen shown to be activated by Wnt signaling (Liu et al., 2004, PNAS,101:4158-4163). In human breast cancer, β-catenin accumulationimplicates activated Wnt signaling in over 50% of carcinomas, and thoughspecific mutations have not been identified, upregulation of Frizzledreceptor expression has been observed (Brennan & Brown, 2004, J. MammaryGland Neoplasia, 9:119-31; Malovanovic et al., 2004, Int. J. Oncol.,25:1337-42).

Colorectal cancer is most commonly initiated by activating mutations inthe Wnt signaling cascade. Approximately 5-10% of all colorectal cancersare hereditary with one of the main forms being familial adenomatouspolyposis (FAP), an autosomal dominant disease in which about 80% ofaffected individuals contain a germline mutation in the adenomatouspolyposis coli (APC) gene. Mutations have also been identified in otherWnt pathway components including Axin and β-catenin. Individual adenomasare clonal outgrowths of epithelial cells containing a secondinactivated allele, and the large number of FAP adenomas inevitablyresults in the development of adenocarcinomas through additionalmutations in oncogenes and/or tumor suppressor genes. Furthermore,activation of the Wnt signaling pathway, including gain-of-functionmutations in APC and β-catenin, can induce hyperplastic development andtumor growth in mouse models (Oshima et al., 1997, Cancer Res,.57:1644-9; Harada et al., 1999, EMBO J., 18:5931-42).

BRIEF SUMMARY OF THE INVENTION

The present invention provides a variety of agents that bind to one ormore human Wnt proteins, including, but not limited to, soluble FZDreceptors and other agents comprising a Fri domain, and novel methods ofusing those agents. The invention further provides methods of using theagents in the treatment of cancer by administering the agents to asubject in need thereof. In some embodiments, the methods compriseinhibiting the growth of cancer cells. In certain embodiments, theWnt-binding agent is a Wnt antagonist. Novel methods of screening forsuch Wnt-binding agents are also provided. Polynucleotides encoding theagents, methods of making the agents, and a variety of compositionscomprising the agents are likewise provided.

Thus, in one aspect, the invention provides a method of inhibiting thegrowth of a tumor. In certain embodiments, the method comprisescontacting the tumor with an effective amount of an agent that binds toone or more Wnt proteins (e.g., human Wnt proteins). The method may bein vivo or in vitro. In certain embodiments, the tumor is in a subject,and the contacting of the tumor with the agent comprises administrationof a therapeutically effective amount of the Wnt-binding agent to thesubject.

In another aspect, the invention provides a method of reducing thefrequency of cancer stem cells in a tumor comprising cancer stem cells.Accordingly, the invention also provides methods of reducing thetumorigenicity of tumors. In some embodiments, the methods comprisecontacting the tumor with an effective amount of an agent that binds toone or more Wnt proteins (e.g., human Wnt proteins). The method may bein vivo or in vitro. For example, the contacting may compriseadministration of the effective amount of the Wnt-binding agent to ahuman having the tumor.

In another aspect, the invention provides a method of inducing cells ina tumor to differentiate or inducing expression of differentiationmarkers in a tumor. In certain embodiments, the method comprisescontacting the tumor with an effective amount of an agent that binds toone or more Wnt proteins (e.g., human Wnt proteins). The method may bein vivo or in vitro.

In a still further aspect, the invention provides a method of treating acancer in a subject, comprising administering a therapeuticallyeffective amount of the Wnt-binding agent to the subject.

In an additional aspect, the invention provides a method of treating adisease in a subject wherein the disease is associated with Wntsignaling activation, comprising administering a therapeuticallyeffective amount of the Wnt-binding agent to the subject.

In yet another aspect, the invention provides a method of treating adisorder in a subject, wherein the disorder is characterized by anincreased level of stem cells and/or progenitor cells, comprisingadministering a therapeutically effective amount of the Wnt-bindingagent to the subject.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the Wnt-binding agent is a polypeptide.In certain embodiments, the agent is a soluble receptor.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the agent comprises a Fri domain of a FZDreceptor, or a fragment of a FZD Fri domain that binds one or more Wntproteins. In certain embodiments, the FZD receptors are human FZDreceptors. For example, the Fri domain may be from human FZD4 or humanFZD5. In certain alternative embodiments, the agent may comprise a Fridomain of a human FZD8 receptor or a fragment of that Fri domain thatbinds one or more Wnt proteins. In certain embodiments, the Wnt-bindingagent is a soluble FZD receptor. In alternative embodiments, theWnt-binding agent does not comprise a Fri domain of a FZD receptor.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the agent comprises a Fri domain of asoluble Frizzled-related protein (SFRP), or a fragment of a SFRP Fridomain that binds one or more Wnt proteins. In certain embodiments theSFRP is a human SFRP.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the agent comprises a Fri domain of a Rorprotein or a fragment of a Ror Fri domain that binds one or more Wntproteins. In certain embodiments the Ror protein is a human Ror protein.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the agent further comprises a human Fcregion. In certain embodiments, the Wnt-binding agent is a fusionprotein. In certain embodiments, the Wnt-binding agent comprises SEQ IDNO:1. In certain embodiments, the Wnt-binding agent comprises SEQ IDNO:46. In certain embodiments, the Wnt-binding agent comprises SEQ IDNO:48. In certain embodiments, the Wnt-binding agent comprises SEQ IDNO:50. In certain embodiments, the Wnt-binding agent comprises SEQ IDNO:53. In some embodiments, the Wnt-binding agent (before signalsequence cleavage) comprises SEQ ID NO:50 and a signal sequence selectedfrom the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ IDNO:74. In some embodiments, the Wnt-binding agent (before signalsequence cleavage) comprises SEQ ID NO:50 and a signal sequence selectedfrom the group consisting of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:73, and SEQ ID NO:74. In some embodiments, the Wnt-bindingagent (before signal sequence cleavage) comprises SEQ ID NO:50 and SEQID NO:71. In some embodiments, the Wnt-binding agent (before signalsequence cleavage) comprises SEQ ID NO:53 and a signal sequence selectedfrom the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ IDNO:74. In some embodiments, the Wnt-binding agent (before signalsequence cleavage) comprises SEQ ID NO:53 and a signal sequence selectedfrom the group consisting of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72,SEQ ID NO:73, and SEQ ID NO:74. In some embodiments, the Wnt-bindingagent (before signal sequence cleavage) comprises SEQ ID NO:53 and SEQID NO:71.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the Wnt-binding agent binds to one ormore, two or more, three or more, or four or more human Wnt proteinsselected from the group consisting of Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a,Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt10a, and Wnt10b. In certain embodiments,the agent binds to Wnt1, Wnt2, Wnt3, Wnt3a, and Wnt7b.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the agent is a Wnt antagonist. In certainembodiments, the agent inhibits Wnt signaling. In certain embodiments,the agent inhibits Wnt canonical Wnt signaling.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the tumor or cancer is a tumor/cancerselected from the group consisting of colorectal tumor/cancer,pancreatic tumor/cancer, lung tumor/cancer, ovarian tumor/cancer, livertumor/cancer, breast tumor/cancer, kidney tumor/cancer, prostatetumor/cancer, gastrointestinal tumor/cancer, melanoma, cervicaltumor/cancer, bladder tumor/cancer, glioblastoma, and head and necktumor/cancer.

In certain embodiments of each of the aforementioned aspects, as well asother aspects provided herein, the methods further comprise contactingthe tumor with a second therapeutic agent or administering a secondtherapeutic agent to the subject. In certain embodiments, the secondtherapeutic agent is a chemotherapeutic agent. In certain embodiments,the second chemotherapeutic agent is an antimetabolite (e.g.,gemcitabine) or an antimitotic agent (e.g., a taxane such aspaclitaxel).

In a still further aspect, the invention provides a polypeptidecomprising a sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49,SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,SEQ ID NO:65 and SEQ ID NO:66, as well as cells producing andcompositions comprising the polypeptide. Pharmaceutical compositionscomprising the polypeptide and a pharmaceutically acceptable carrier arealso provided. In addition, polynucleotides comprising a polynucleotidethat encodes the polypeptides of SEQ ID NO:1, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:65 and SEQ IDNO:66 or having the sequence of SEQ ID NO:2 are also provided. Vectorsand cells comprising the polynucleotides are likewise provided.

In an additional aspect, the invention provides a method of screening anagent for anti-tumor activity and/or activity against cancer stem cells.In certain embodiments, the method comprises comparing the level of oneor more differentiation markers and/or one or more stemness markers in afirst solid tumor (e.g., a solid tumor comprising cancer stem cells)that has been exposed to an agent to the level of the one or moredifferentiation markers in a second solid tumor that has not beenexposed to the agent. In some embodiments, the method comprises: (a)exposing a first solid tumor, but not a second solid tumor, to theagent; (b) assessing the level of one or more differentiation markersand/or one or more stemness markers in the first and second solidtumors; and (c) comparing the level of the one or more differentiationmarkers in the first tumor to the level of the one or moredifferentiation markers in the second solid tumor. In certainembodiments, the (a) increased levels of the one or more differentiationmarkers in the first solid tumor relative to the second solid tumorindicates anti-tumor or anti-cancer stem cell activity of the agent;and/or (b) decreased levels of the one or more sternness markersindicates anti-tumor or anti-cancer stem cell activity of the agent. Incertain embodiments, the agent binds one or more Wnt proteins. Incertain embodiments, the agent is a soluble FZD receptor. In certainmethods, the agent is an antibody, such as an anti-FZD or anti-Wntantibody. In certain alternative embodiments, the agent is a smallmolecule.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Pharmacokinetics of FZD8-Fc (54F03) in the rat. Administrationof a single dose (10mg/kg) of FZD8-Fc was followed by assessment of thepharmacokinetic properties of FZD8-Fc. Serum concentrations of FZD8-Fcwere determined at 1, 24, 48, 72, 96, 168, 240 and 336 hourspost-administration.

FIG. 2. Inhibition of PN4 pancreas tumor growth by FZD8-Fc (54F03)treatment. PN4 pancreas tumor cells were injected subcutaneously intoNOD/SCID mice. Mice were treated with FZD8-Fc (-▴-), gemcitabine (-▪-),a combination of FZD8-Fc and gemcitabine (-Δ-), or a control antibody(-•-). Data are shown as tumor volume (mm³), over days post treatment.

FIG. 3. Reduction of CD44^(hi) cell population in PN4 tumors treatedwith FZD8-Fc (54F03). Cell surface staining for ESA and CD44 on tumorcells treated with control antibody, FZD8-Fc, gemcitabine, or acombination of FZD8-Fc and gemcitabine was performed. For each treatmentgroup, single cell suspensions from five tumors were pooled prior tostaining.

FIG. 4. In vivo limiting dilution assay of FZD8-Fc (54F03)-treated PN4pancreatic tumors.

FIG. 5. Increased cell differentiation of PN4 pancreatic tumors treatedwith FZD8-Fc (54F03). Paraffin embedded sections of PN4 tumors treatedwith control antibody, FZD8-Fc, gemcitabine, or a combination of FZD8-Fcand gemcitabine were stained with alcian blue to detect mucin-expressingcells.

FIG. 6. Increased cell differentiation of PN8 pancreatic tumors treatedwith FZD8-Fc (54F03). Paraffin embedded sections of PN8 tumors treatedwith control antibody, FZD8-Fc, gemcitabine, or a combination of FZD8-Fcand gemcitabine were stained with alcian blue to detect mucin-expressingcells.

FIG. 7. Increased cell differentiation and reduced proliferation in PN13pancreatic tumors following treatment with FZD8-Fc (54F03). Paraffinembedded sections of PN13 tumors treated with control antibody orFZD8-Fc were stained with alcian blue to detect mucin-expressing cells.In addition, the sections were stained for Ki67, a marker of activelyproliferating cells.

FIG. 8. Increased Mucl6 staining in PN13 pancreatic tumors followingtreatment with FZD8-Fc (54F03). Paraffin embedded sections of PN13tumors treated with a control antibody or FZD8-Fc were stained for Muc16 protein.

FIG. 9. Increased CK20 staining in PN13 pancreatic tumors followingtreatment with FZD8-Fc (54F03). Paraffin embedded sections of PN13tumors treated with a control antibody or FZD8-Fc were stained for CK20protein.

FIG. 10 Inhibition of PE13 breast tumor growth following treatment withFZD8-Fc (54F03) in combination with paclitaxel. PE13 breast tumor cellswere injected subcutaneously into NOD/SCID mice. Mice were treated witha control antibody (-•-), FZD8-Fc (□), paclitaxel (-▴-), or acombination of FZD8-Fc and paclitaxel (-∘-). Data is shown as tumorvolume (mm³) over days post treatment.

FIG. 11. Dose-dependent inhibition of C28 colon tumor growth withFZD8-Fc (54F03). C28 colon tumor cells were injected subcutaneously intoNOD/SCID mice. Mice were treated with FZD8-Fc 1.5 mg/kg twice weekly(-▴-), 5 mg/kg once weekly (-▾-), 5 mg/kg twice weekly (-∘-),15 mg/kgonce weekly (-□-) or 15 mg/kg twice weekly (-Δ-) or control antibody(-▪-). Data is shown as tumor volume (mm³) over days post treatment(FIG. 11A) Inhibition of colon tumor growth with FZD8-Fc in combinationwith irinotecan. Mice were treated with FZD8-Fc (-▪-), irinotecan (-▾-),a combination of FZD8-Fc and irinotecan (-•-), or a control antibody(-▪-). Data is shown as tumor volume (mm³) over days post treatment(FIG. 11B).

FIG. 12. Increased CK20 staining in C28 tumors following treatment withFZD8-Fc (54F03). Paraffin embedded sections of C28 tumors treated with acontrol antibody or FZD8-Fc were stained for CK20 protein.

FIG. 13 Inhibition of PN21 pancreatic tumor growth and decrease incancer stem cell frequency by FZD8-Fc (54F03) treatment. PN21 pancreatictumor cells were injected subcutaneously into NOD/SCID mice. Mice weretreated with FZD8-Fc (-▾-), gemcitabine (-▴-), a combination of FZD8-Fcand gemcitabine (-▪-), or a control antibody (-•-). Data are shown astumor volume (mm³), over days post treatment (FIG. 13A). In vivolimiting dilution assay of FZD8-Fc treated PN21 pancreatic tumors (FIG.13B).

FIG. 14. Increased cell differentiation of PN21 pancreatic tumorsfollowing treatment with FZD8-Fc (54F03). Paraffin embedded sections ofPN21 tumors treated with control antibody or FZD8-Fc were stained withalcian blue to detect mucins.

FIG. 15. Increased cell differentiation and reduced proliferation inPN21 pancreatic tumors following treatment with FZD8-Fc (54F03).Paraffin embedded sections of PN21 tumors treated with control antibody,FZD8-Fc, gemcitabine, or a combination of FZD8-Fc and gemcitabine werestained with alcian blue to detect mucins. In addition, the sectionswere stained for Ki67, a marker of actively proliferating cells.

FIG. 16. Pharmacokinetics of FZD8-Fc variants in monkeys. Administrationof a single dose (30 mg/kg) of FZD8-Fc variants 54F15 and 54F16 wasfollowed by assessment of the pharmacokinetic properties of thevariants. Serum concentrations of 54F15 (-∘-) and 54F16 (-♦-) weredetermined at 1, 6, 12, 24, 48, 72, 96, 168, 240 and 336 hourspost-administration.

FIG. 17. Inhibition of C28 colon tumor growth following treatment withFZD8-Fc variants. C28 colon tumor cells were injected subcutaneouslyinto NOD/SCID mice. Mice were treated with a control antibody (-X-),54F03 (-□-), 54F09 (-▾-), 54F12 (-∘-), 54F13 (-♦-), 54F15 (-∘-) or 54F16(-□-). Data is shown as tumor volume (mm³) over days post treatment.

FIG. 18 Inhibition of PN4 pancreatic tumor growth following treatmentwith FZD8-Fc variants. PN4 colon tumor cells were injectedsubcutaneously into NOD/SCID mice. Mice were treated with a controlantibody (-•-), 54F03 (-▪-), 54F09 (-▾-), 54F12 (-∘-), 54F13 (-▴-),54F15 (-□-) or 54F16 (-♦-). Data is shown as tumor volume (mm³) overdays post treatment.

FIG. 19. Reduction of CD44^(hi) and CD44⁺CD201⁺ cells in PN4 tumorstreated with FZD8-Fc variants 54F03 and 54F16. Cell surface staining forESA, CD44 and CD201 on tumor cells treated with control antibody,FZD8-Fc variant 54F03 or variant 54F16 was performed and analyzed byFACS.

FIG. 20. Characterization of N-termini of FZD8-Fc proteins. FZD8-Fcvariants were analyzed by mass spectrometry and results for 54F16 (FIG.20A), 54F26 (FIG. 20B), 54F28 (FIG. 20C), 54F30 (FIG. 20D), and 54F32(FIG. 20E) are shown.

FIG. 21. Inhibition of C28 colon tumor growth following treatment withFZD8-Fc variants. C28 colon tumor cells were injected subcutaneouslyinto NOD/SCID mice. Mice were treated with a control antibody (-▪-),54F03 (-Δ-), 54F23 (-▾-), or 54F26 (-∘-). Data is shown as tumor volume(mm³) over days post treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limitedto, polypeptides comprising the Fri domain of human Frizzled (FZD)receptors, human secreted Frizzled-related proteins (SFRPs), or Rorproteins that bind to one or more human Wnts. Related polypeptides andpolynucleotides, compositions comprising the Wnt-binding agents, andmethods of making the Wnt-binding agents are also provided. Methods ofusing the Wnt-binding agents, such as methods of inhibiting tumorgrowth, treating cancer, inducing differentiation, and reducingtumorigenicity are further provided. Methods of screening agents toidentify novel Wnt-binding agents with anti-tumor activity and/oranti-cancer stem cell activity are also provided.

A Wnt-binding agent comprising the Fri domain of human FZD8 and a Fcdomain was produced and referred to herein as FZD8-Fc or FZD8-Fc (54F03)(Example 1). A number of variants of the FZD8-Fc protein were generated(Example 10). FZD8-Fc proteins were produced that were shown to beapproximately 95% or greater homogeneous at the N-termini (Example 16).Pharmacokinetic studies using several of the FZD8-Fc variants were donein rats showing that the half-life of the FZD8-Fc variants was at least100 hours (Examples 2 and 12; FIG. 1 and Table 4). A pharmacokineticstudy was undertaken in monkeys with FZD8-Fc variants 54F15 and 54F16,which demonstrated that these proteins had a half-life of at least 100hours (Example 13 and Table 5). Treatment with FZD8-Fc (54F03), eitheralone or in combination with a chemotherapeutic agent was shown toreduce the growth of pancreatic tumors, breast tumors and colon tumors(Examples 3, 5, 6 and 8 and FIGS. 2, 10, 11B, and 13A). Furthermore, thetreatment was shown to decrease the percentage of CD44⁺ cells and toreduce the frequency of cancer stem cells in the pancreatic model(Examples 3 and 8 and FIGS. 3, 4, and 13B). Treatment with FZD8-Fc(54F03), either alone or in combination with a chemotherapeutic agentwas shown to increase cell differentiation of pancreatic tumor cells andcolon tumor cells (Examples 4, 7 and 9 and FIGS. 5-9, 12, 14, and 15).Treatment with FZD8-Fc variants demonstrated inhibition of tumor growthin colon and pancreatic tumors, with the extent of inhibition dependingupon the variant (Examples 14, 15, and 17 and FIGS. 17, 18, and 21).Treatment with FZD8-Fc variant 54F03 and variant 54F16 was shown todecrease the percentage of CD44^(hi) cells, as well as CD44⁺CD202⁺ cellsin pancreatic tumors (Example 15 and FIG. 19).

I. Definitions

The term “antagonist” is used herein to include any molecule thatpartially or fully blocks, inhibits, or neutralizes the expression of orthe biological activity of a protein, (e.g., a cancer stem cell marker).The blocking, inhibiting, and/or neutralizing of biological activityincludes, but is not limited to, inhibition of tumor growth. The term“antagonist” includes any molecule that partially or fully blocks,inhibits, or neutralizes a biological activity of the Wnt pathway.Suitable antagonist molecules include, but are not limited to, fragmentsand/or amino acid sequence variants of native FZD receptor proteinsincluding soluble FZD receptors, as well as derivatives of SFRPs andderivatives of Ror proteins.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cells orcompositions include those which have been purified to a degree thatthey are no longer in a form in which they are found in nature. In someembodiments, an antibody, polynucleotide, vector, cell, or compositionwhich is isolated is substantially pure.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably at least90% pure, more preferably at least 95% pure, more preferably at least98% pure, more preferably at least 99% pure.

As used herein the term “soluble receptor” refers to an N-terminalextracellular fragment of a receptor protein preceding the firsttransmembrane domain of the receptor that can be secreted from a cell insoluble form. In some embodiments, the receptor protein is a FZDreceptor. In some embodiments, the receptor protein is a Ror receptor.

As used herein the term “FZD soluble receptor” refers to an N-terminalextracellular fragment of a human FZD receptor protein preceding thefirst transmembrane domain of the receptor that can be secreted from acell in soluble form. Both FZD soluble receptors comprising the entireN-terminal extracellular domain (ECD) (referred to herein as “FZD ECD”)as well as smaller fragments are envisioned. FZD soluble receptorscomprising the Fri domain (referred to herein as “FZD Fri”) are alsodisclosed. FZD Fri soluble receptors can demonstrate altered biologicalactivity, (e.g., increased protein half-life) compared to solublereceptors comprising the entire FZD ECD. Protein half-life can befurther increased by covalent modification with polyethylene glycol(PEG) or polyethylene oxide (PEO). FZD soluble receptors include FZD ECDor Fri domains linked in-frame to other functional and structuralproteins including, but not limited to, a human Fc region (e.g., humanFc derived from immunoglobulins IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD,IgE, or IgM); protein tags (e.g., myc, FLAG, GST); other endogenousproteins or protein fragments; or any other useful protein sequenceincluding any linker region between a FZD ECD or Fri domain and a linkedprotein. In certain embodiments, the Fri domain of a FZD receptor isdirectly linked to a human Fc region. In certain embodiments, the Fridomain of a FZD receptor is linked to human IgG1 Fc (referred to hereinas “FZD Fri.Fc”). In some embodiments, the Fri domain of a FZD receptoris linked to a human Fc region with a peptide linker FZD solublereceptors also include variant proteins comprising amino acidinsertions, deletions, substitutions, and/or conservative substitutions.

As used herein, the term “linker” or “linker region” refers to a linkerinserted between a first polypeptide (e.g., a FZD component) and asecond polypeptide (e.g., a Fc region). In some embodiments, the linkeris a peptide linker Linkers should not adversely affect the expression,secretion, or bioactivity of the polypeptides. Preferably, linkers arenot antigenic and do not elicit an immune response.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals in which a population of cellsare characterized by unregulated cell growth. The term cancer isunderstood to encompass Wnt-dependent cancers. Examples of cancersinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia. More particular examples of such cancers include squamouscell cancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, skin cancer, melanoma, endometrial or uterinecarcinoma, salivary gland carcinoma, kidney cancer, liver cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma andvarious types of head and neck cancer.

The terms “proliferative disorder” and “proliferative disease” refer todisorders associated with abnormal cell proliferation such as cancer.

“Tumor” and “neoplasm” as used herein refer to any mass of tissue thatresults from excessive cell growth or proliferation, either benign(noncancerous) or malignant (cancerous) including pre-cancerous lesions.In certain embodiments, the tumor is an epithelial tumor. In certainembodiments, the tumor is a Wnt-dependent tumor.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,canines, felines, rodents, and the like, which is to be the recipient ofa particular treatment. Typically, the terms “subject” and “patient” areused interchangeably herein in reference to a human subject.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “solidtumor stem cell” are used interchangeably herein and refer to apopulation of cells from a solid tumor that: (1) have extensiveproliferative capacity; (2) are capable of asymmetric cell division togenerate one or more kinds of differentiated progeny with reducedproliferative or developmental potential; and (3) are capable ofsymmetric cell divisions for self-renewal or self-maintenance. Theseproperties of “cancer stem cells,” “CSCs,” “tumor stem cells,” or “solidtumor stem cells” confer on those cancer stem cells the ability to formpalpable tumors upon serial transplantation into an immunocompromisedhost (e.g., a mouse) compared to the majority of tumor cells that failto form tumors. Cancer stem cells undergo self-renewal versusdifferentiation in a chaotic manner to form tumors with abnormal celltypes that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” and grammatical equivalentsrefer to the total population of cells derived from a tumor includingboth non-tumorigenic cells, which comprise the bulk of the tumor cellpopulation, and tumorigenic stem cells also referred to herein as cancerstem cells.

The term “tumorigenic” refers to the functional features of a solidtumor stem cell including the properties of self-renewal (giving rise toadditional tumorigenic cancer stem cells) and proliferation to generateall other tumor cells (giving rise to differentiated and thusnon-tumorigenic tumor cells) that allow solid tumor stem cells to form atumor. These properties of self-renewal and proliferation to generateall other tumor cells confer on cancer stem cells the ability to formpalpable tumors upon serial transplantation into an immunocompromisedhost (e.g., a mouse) compared to non-tumorigenic tumor cells, which areunable to form tumors upon serial transplantation. It has been observedthat non-tumorigenic tumor cells may form a tumor upon primarytransplantation into an immunocompromised host (e.g., a mouse) afterobtaining the tumor cells from a solid tumor, but those non-tumorigenictumor cells do not give rise to a tumor upon serial transplantation.

As used herein an “acceptable pharmaceutical carrier” or“pharmaceutically acceptable carrier” refers to any material that, whencombined with an active ingredient of a pharmaceutical composition suchas a therapeutic polypeptide, allows the therapeutic polypeptide, forexample, to retain its biological activity. In addition, an “acceptablepharmaceutical carrier” does not trigger an immune response in arecipient subject. In some embodiments, the term “pharmaceuticalvehicle” is used interchangeably with “pharmaceutical carrier”. Examplesinclude, but are not limited to, any of the standard pharmaceuticalcarriers such as a phosphate buffered saline solution, water, andvarious oil/water emulsions. Examples of diluents for aerosol orparenteral administration are phosphate buffered saline or normal (0.9%)saline.

The term “therapeutically effective amount” refers to an amount of anagent (e.g., a soluble receptor or other drug) effective to “treat” adisease or disorder in a subject or mammal. In the case of cancer, thetherapeutically effective amount of the agent (e.g., a soluble receptor)can reduce the number of cancer cells; reduce the tumor size; reduce thefrequency of cancer stem cells; inhibit and/or stop cancer cellinfiltration into peripheral organs; inhibit and/or stop tumormetastasis; inhibit and/or stop tumor growth; and/or relieve to someextent one or more of the symptoms associated with the cancer. To theextent the agent (e.g., a soluble receptor) prevents growth and/or killsexisting cancer cells, it can be referred to as cytostatic and/orcytotoxic.

As used herein the term “inhibit tumor growth” refers to any mechanismby which tumor cell growth can be inhibited. In certain embodiments,tumor cell growth is inhibited by slowing proliferation of tumor cells.In certain embodiments, tumor cell growth is inhibited by haltingproliferation of tumor cells. In certain embodiments, tumor cell growthis inhibited by killing tumor cells. In certain embodiments, tumor cellgrowth is inhibited by inducing apoptosis of tumor cells. In certainembodiments, tumor cell growth is inhibited by inducing differentiationof tumor cells. In certain embodiments, tumor cell growth is inhibitedby depriving tumor cells of nutrients. In certain embodiments, tumorcell growth is inhibited by preventing migration of tumor cells. Incertain embodiments, tumor cell growth is inhibited by preventinginvasion of tumor cells.

Terms such as “treating” and “treatment” and “to treat” and“alleviating” and “to alleviate” refer to both 1) therapeutic measuresthat cure, slow down, lessen symptoms of, and/or halt progression of adiagnosed pathologic condition or disorder, and 2) prophylactic orpreventative measures that prevent or slow the development of a targetedpathologic condition or disorder. Thus, those in need of treatmentinclude those who already have the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented. In certainembodiments, a subject is successfully “treated” for cancer according tothe methods of the present invention if the patient shows one or more ofthe following: a reduction in the number of, or complete absence of,cancer or tumor cells; a reduction in the tumor size; inhibition of, oran absence of, cancer or tumor cell infiltration into peripheral organsincluding, for example, the spread of cancer into soft tissue and bone;inhibition of, or an absence of, tumor metastasis; inhibition of, or anabsence of, tumor or cancer growth; relief of one or more symptomsassociated with the specific cancer; reduced morbidity and mortality;improvement in quality of life; reduction in tumorigenicity, tumorgenicfrequency, or tumorgenic capacity of a tumor; reduction in the number orfrequency of cancer stem cells in the tumor; differentiation oftumorigenic cells to a non-tumorigenic state; or some combination ofthese effects.

As used herein, the terms “polynucleotide” and “nucleic acid” refer to apolymer composed of a multiplicity of nucleotide units (ribonucleotideor deoxyribonucleotide or related structural variants) linked viaphosphodiester bonds, including but not limited to, DNA or RNA. The termencompasses sequences that include any of the known base analogs of DNAand RNA including, but not limited to, 4-acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.Other types of modifications include, for example, “caps”; substitutionof one or more of the naturally occurring nucleotides with an analog;internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) andcharged linkages (e.g., phosphorothioates, phosphorodithioates, etc.);pendant moieties, such as proteins (e.g., nucleases, toxins, antibodies,signal peptides, poly-L-lysine, etc.); intercalators (e.g., acridine,psoralen, etc.); chelators (e.g., metals, radioactive metals, boron,oxidative metals, etc.); alkylators; modified linkages (e.g., alphaanomeric nucleic acids, etc.); as well as unmodified forms of thepolynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, heptuloses, acyclic analogsand abasic nucleoside analogs such as methyl riboside. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S(“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vector” means a construct, which is capable of delivering, andpreferably expressing, one or more gene(s) or sequence(s) of interest toa host cell. Examples of vectors include, but are not limited to, viralvectors, naked DNA or RNA expression vectors, plasmid, phagemid, cosmidor phage vectors, DNA or RNA expression vectors associated with cationiccondensing agents, and DNA or RNA expression vectors encapsulated inliposomes.

The terms “polypeptide” and “peptide” and “protein” and “proteinfragment” are used interchangeably herein to refer to a polymer of aminoacid residues of any length. The terms apply to amino acid polymers inwhich one or more amino acid residue in the polymer is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymers. The polymer may be linear or branched, itmay comprise modified amino acids, and it may be interrupted bynon-amino acids. The terms also encompass an amino acid polymer that hasbeen modified naturally or by intervention; for example, disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation or modification, such as conjugation with alabeling component. Also included within the definition are, forexample, polypeptides containing one or more analogs of an amino acid(including, for example, unnatural amino acids, etc.), as well as othermodifications known in the art. It is understood that, because thepolypeptides of this invention are based, at least in part, uponantibodies, in certain embodiments, the polypeptides can occur as singlechains or associated chains.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid Amino acid mimetic refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that function similarly to anaturally occurring amino acid.

That a polypeptide or other agent “specifically binds” to a proteinmeans that the polypeptide or other agent reacts or associates morefrequently, more rapidly, with greater duration, with greater affinity,or with some combination of the above to the protein than withalternative substances, including unrelated proteins. In certainembodiments, “specifically binds” means, for instance, that an agentbinds to a protein with a K_(D) of about 0.1 mM or less, but moreusually less than about 1 μM. In certain embodiments, “specificallybinds” means that an agent binds to a protein at times with a K_(D) ofat least about 0.1 μM or less, at least about 0.01 μM or less, and atother times at least about 1 nM or less. Because of the sequenceidentity between homologous proteins in different species, specificbinding can include an agent that recognizes a particular protein suchas a Wnt protein in more than one species. Likewise, because of homologybetween different Wnt proteins in certain regions of the sequences ofthe Wnts, specific binding can include an polypeptide (or other agent)that recognizes more than one Wnt protein. It is understood that anagent that specifically binds to a first target may or may notspecifically bind to a second target. As such, “specific binding” doesnot necessarily require (although it can include) exclusive binding,i.e. binding to a single target. Thus, an agent may, in certainembodiments, specifically bind to more than one target (e.g., multipledifferent human Wnts). Generally, but not necessarily, reference tobinding means specific binding.

The terms “identical” or “percent identity” in the context of two ormore nucleic acids or polypeptides, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity may be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software that may be used to obtain alignments of aminoacid or nucleotide sequences are known in the art. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified inKarlin et al., 1993, PNAS, 90:5873-5877, and incorporated into theNBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res.,25:3389-3402). Additional publicly available software programs that canbe used to align sequences include, but are not limited to, GappedBLAST, BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South SanFrancisco, Calif.), Megalign (DNASTAR), and the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711).

In some embodiments, two nucleic acids or polypeptides of the inventionare substantially identical, meaning they have at least 70%, at least75%, at least 80%, at least 85%, at least 90%, and in some embodimentsat least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residueidentity, when compared and aligned for maximum correspondence, asmeasured using a sequence comparison algorithm or by visual inspection.In some embodiments, identity exists over a region of the sequences thatis at least about 10, at least about 20, at least about 40-60, at leastabout 60-80 residues in length or any integral value therebetween. Insome embodiments, identity exists over a longer region than 60-80residues, such as at least about 90-100 residues. In some embodiments,the sequences are substantially identical over the full length of thesequences being compared, such as the coding region of a nucleotidesequence.

A “conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). For example, substitution of aphenylalanine for a tyrosine is a conservative substitution. Preferably,conservative substitutions in the sequences of the polypeptides andother agents of the invention do not abrogate the binding of thepolypeptide containing the amino acid sequence, to the target(s), i.e.,the one or more Wnts to which the polypeptide or other agent binds.Methods of identifying nucleotide and amino acid conservativesubstitutions which do not eliminate target binding are well-known inthe art (see, e.g., Brummell et al., 1993, Biochem., 32: 1180-87;Kobayashi et al., 1999, Protein Eng. 12:879-84; and Burks et al., 1997,PNAS, 94:412-17).

As used herein, “about” refers to plus or minus 10% of the indicatednumber. For example, “about 10%” indicates a range of 9% to 11%.

As used in the present disclosure and claims, the singular forms “a”“an” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone), and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A(alone); B (alone); and C (alone).

II. Wnt-Binding Agents

The present invention provides agents that bind (e.g., specificallybind) one or more human Wnt proteins (Wnts). These agents are referredto herein as “Wnt-binding agent(s).” In certain embodiments, the agentsspecifically bind one, two, three, four, five, six, seven, eight, nine,ten, or more Wnt proteins. By way of non-limiting example, theWnt-binding agent may bind Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b,Wnt8a, Wnt8b, Wnt10a, and/or Wnt10b. In certain embodiments, theWnt-binding agent binds Wnt1, Wnt2, Wnt3, Wnt3a, and Wnt7b.

In certain embodiments, the Wnt-binding agent is a Wnt antagonist. Incertain embodiments, the agent inhibits Wnt-signaling. In someembodiments, the agent inhibits canonical Wnt signaling.

In certain embodiments, the Wnt-binding agent is a polypeptide. Incertain embodiments, the Wnt-binding agent is a soluble receptor.

In certain embodiments, the Wnt-binding agent comprises theextracellular domain of a FZD receptor. In some embodiments, theWnt-binding agent comprises a Fri domain of a FZD receptor. In certainembodiments, the FZD receptor is a human FZD receptor. In certainembodiments, the human FZD receptor is FZD1, FZD2, FZD3, FZD4, FZD5,FZD6, FZD7, FZD8, FZD9, or FZD10. In some alternative embodiments, theWnt-binding agent comprises a portion of a SFRP. In some embodiments,the Wnt-binding agent comprises a Fri domain of a SFRP. In certainembodiments, the SFRP is a human SFRP. In some embodiments, the humanSFRP is SFRP1, SFRP2, SFRP3, SFRP4, or SFRP5. In other alternativeembodiments, the Wnt-binding agent comprises the extracellular domain ofa Ror protein. In some embodiments, the Wnt-binding agent comprises aFri domain of a Ror protein. In certain embodiments, the Ror is a humanRor. In some embodiments, the human Ror is Ror1 or Ror2.

In certain embodiments, the Wnt-binding agent is a soluble receptor. Insome embodiments, the Wnt-binding agent is a soluble protein. In certainembodiments, the Wnt-binding agent is a soluble FZD receptor.Nonlimiting examples of soluble FZD receptors can be found in U.S. Pat.No. 7,723,477, which is incorporated by reference herein in itsentirety. In certain embodiments, the Wnt-binding agent is a solubleSFRP or a soluble Ror receptor.

The Fri domain of FZD1 includes approximately amino acids 87-237 of SEQID NO:27. The Fri domain of FZD2 includes approximately amino acids24-159 of SEQ ID NO:28. The Fri domain of FZD3 includes approximatelyamino acids 23-143 of SEQ ID NO:29. The Fri domain of FZD4 includesapproximately amino acids 40-170 of SEQ ID NO:22. The Fri domain of FZD5includes approximately amino acids 27-157 of SEQ ID NO:23. The Fridomain of FZD6 includes approximately amino acids 19-146 of SEQ IDNO:24. The Fri domain of FZD7 includes approximately amino acids 33-170of SEQ ID NO:25. The Fri domain of FZD8 includes approximately aminoacids 28-158 of SEQ ID NO:30. The Fri domain of FZD9 includesapproximately amino acids 23-159 of SEQ ID NO:31. The Fri domain ofFZD10 includes approximately amino acids 21-154 of SEQ ID NO:26. Thecorresponding, predicted Fri domains for each of the human FZD receptorsare provided as SEQ ID NOs:32-41. The minimal, core Fri domain sequencesfor each of the human FZD receptors (FZD1-10) are provided as SEQ IDNOs:3-12. The minimal, core Fri domain sequences for each of the humanSFRPs (SFRP1-5) are provided as SEQ ID NOs:13-17. The minimal, core Fridomain sequences of human Rorl and Ror2 are provided as SEQ ID NO:58 andSEQ ID NO:59. Those of skill in the art may differ in theirunderstanding of the exact amino acids corresponding to the various Fridomains. Thus the N-terminus or C-terminus of the domains outlined aboveand herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, oreven 10 amino acids.

In certain embodiments, the Wnt-binding agent comprises a Fri domain ofa human FZD receptor, or a fragment or variant of the Fri domain thatbinds one or more human Wnt proteins. In certain embodiments, the humanFZD receptor is FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, orFZD10. In certain embodiments, the human FZD receptor is FZD4. Incertain alternative embodiments, the human FZD receptor is FZD5. Incertain additional alternative embodiments, the human FZD receptor isFZD8. In certain embodiments, the FZD is FZD4 and the Wnt-binding agentcomprises SEQ ID NO:6 or comprises approximately amino acids 40 to 170of SEQ ID NO:19. In certain embodiments, the FZD is FZD5 and theWnt-binding agent comprises SEQ ID NO:7 or comprises approximately aminoacids 27-157 of SEQ ID NO:20. In certain embodiments, the FZD is FZD7and the Wnt-binding agent comprises SEQ ID NO:9 or comprisesapproximately amino acids 33 to 170 of SEQ ID NO:25. In certainembodiments, the FZD is FZD8 and the Wnt-binding agent comprises SEQ IDNO:10 or comprises approximately amino acids 28-158 of SEQ ID NO:21. Incertain embodiments, the FZD is FZD10 and the Wnt-binding agentcomprises SEQ ID NO:12 or comprises approximately amino acids 21-154 ofSEQ ID NO:26.

In certain embodiments, the Wnt-binding agent comprises a minimal Fridomain sequence selected from the group consisting of SEQ ID NOs:3-12.In certain embodiments, the Wnt-binding agent comprises a minimal Fridomain sequence selected from the group consisting of SEQ ID NOs:13-17.In certain embodiments, the Wnt-binding agent comprises a minimal Fridomain sequence selected from the group consisting of SEQ ID NO:58 andSEQ ID NO:59.

In certain embodiments, the Wnt-binding agent comprises a variant of anyone of the aforementioned FZD Fri domain sequences that comprises one ormore (e.g., one, two, three, four, five, six, seven, eight, nine, ten,etc.) conservative substitutions and is capable of binding Wnt(s).

In certain alternative embodiments, the Wnt-binding agent comprises aFri domain of a human SFRP, or a fragment or variant of such a Fridomain that binds to one or more human Wnt proteins. For example, incertain embodiments, the agent comprises a minimal SFRP Fri domainsequence selected from the group consisting of SEQ ID NOs:13-17. Incertain embodiments, the Wnt-binding agent comprises a variant of anyone of the aforementioned SFRP Fri domain sequences that comprises oneor more (e.g., one, two, three, four, five, six, seven, eight, nine,ten, etc.) conservative substitutions and maintains the ability to bindWnt(s).

In certain alternative embodiments, the Wnt-binding agent comprises aFri domain of a human Ror protein, or a fragment or variant of such aFri domain that binds to one or more human Wnt proteins. For example, incertain embodiments, the agent comprises a minimal Ror Fri domainsequence selected from the group consisting of SEQ ID NO:58 and SEQ IDNO:59. In certain embodiments, the Wnt-binding agent comprises a variantof any one of the aforementioned Ror Fri domain sequences that comprisesone or more (e.g., one, two, three, four, five, six, seven, eight, nine,ten, etc.) conservative substitutions and maintains the ability to bindWnt(s).

In certain embodiments, the Wnt-binding agent, such as an agentcomprising a minimum Fri domain of a human FZD receptor or other solubleFZD receptor, further comprises a human Fc region (e.g., a human IgG1 Fcregion). The Fc region can be obtained from any of the classes ofimmunoglobulin, IgG, IgA, IgM, IgD and IgE. In some embodiments, the Fcregion is a wild-type Fc region. In some embodiments, the Fc region is amutated Fc region. In some embodiments, the Fc region is truncated atthe N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids,(e.g., in the hinge domain). In some embodiments, an amino acid in thehinge domain is changed to hinder undesirable disulfide bond formation.In some embodiments, a cysteine is replaced with a serine to hinderundesirable disulfide bond formation. In certain embodiments, the Fcregion comprises or consists of SEQ ID NO:18, SEQ ID NO:42, or SEQ IDNO:43.

In certain embodiments, a Wnt-binding agent is a fusion proteincomprising at least a minimum Fri domain of a FZD receptor, a SFRP orRor protein and a Fc region. As used herein, a “fusion protein” is ahybrid protein expressed by a nucleic acid molecule comprisingnucleotide sequences of at least two genes. In some embodiments, theC-terminus of the first polypeptide is linked to the N-terminus of theimmunoglobulin Fc region. In some embodiments, the first polypeptide(e.g., a FZD Fri domain) is directly linked to the Fc region (i.e.without an intervening peptide linker). In some embodiments, the firstpolypeptide is linked to the Fc region via a peptide linker.

As used herein, the term “linker” refers to a linker inserted between afirst polypeptide (e.g., a FZD component) and a second polypeptide(e.g., a Fc region). In some embodiments, the linker is a peptide linkerLinkers should not adversely affect the expression, secretion, orbioactivity of the polypeptide. Linkers should not be antigenic andshould not elicit an immune response. Suitable linkers are known tothose of skill in the art and often include mixtures of glycine andserine residues and often include amino acids that are stericallyunhindered. Other amino acids that can be incorporated into usefullinkers include threonine and alanine residues. Linkers can range inlength, for example from 1-50 amino acids in length, 1-22 amino acids inlength, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3amino acids in length. Linkers may include, but are not limited to,SerGly, GGSG, GSGS, GGGS, S(GGS)_(n) where n is 1-7, GRA, poly(Gly),poly(Ala), ESGGGGVT (SEQ ID NO:60), LESGGGGVT (SEQ ID NO:61), GRAQVT(SEQ ID NO:62), WRAQVT (SEQ ID NO:63), and ARGRAQVT (SEQ ID NO:64). Asused herein, a linker is an intervening peptide sequence that does notinclude amino acid residues from either the C-terminus of the firstpolypeptide (e.g., a FZD Fri domain) or the N-terminus of the secondpolypeptide (e.g., the Fc region).

FZD receptors, SFRPs and Ror proteins contain a signal sequence thatdirects the transport of the proteins. Signal sequences (also referredto as signal peptides or leader sequences) are located at the N-terminusof nascent polypeptides. They target the polypeptide to the endoplasmicreticulum and the proteins are sorted to their destinations, forexample, to the inner space of an organelle, to an interior membrane, tothe cell's outer membrane, or to the cell exterior via secretion. Mostsignal sequences are cleaved from the protein by a signal peptidaseafter the proteins are transported to the endoplasmic reticulum. Thecleavage of the signal sequence from the polypeptide usually occurs at aspecific site in the amino acid sequence and is dependent upon aminoacid residues within the signal sequence. Although there is usually onespecific cleavage site, more than one cleavage site may be recognizedand/or used by a signal peptidase resulting in a non-homogenousN-terminus of the polypeptide. For example, the use of differentcleavage sites within a signal sequence can result in a polypeptideexpressed with different N-terminal amino acids. Accordingly, in someembodiments, the polypeptides as described herein may comprise a mixtureof polypeptides with different N-termini. In some embodiments, theN-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In someembodiments, the polypeptide is substantially homogeneous, i.e., thepolypeptides have the same N-terminus. In some embodiments, the signalsequence of the polypeptide comprises one or more (e.g., one, two,three, four, five, six, seven, eight, nine, ten, etc.) amino acidsubstitutions and/or deletions. In some embodiments, the signal sequenceof the polypeptide comprises amino acid substitutions and/or deletionsthat allow one cleavage site to be dominant, thereby resulting in asubstantially homogeneous polypeptide with one N-terminus. In someembodiments, the signal sequence is SEQ ID NO:67 (amino acids 1-27 ofSEQ ID NO:30). In some embodiments, amino acids 25 and/or 26 of SEQ IDNO:67 are substituted with different amino acids. In some embodiments,amino acids 17, 18, 19, 23, 24, 25, and/or 26 of SEQ ID NO:67 aresubstituted with different amino acids. In some embodiments, amino acids17, 23, 24, 25, and 26 of SEQ ID NO:67 are substituted with differentamino acids. In some embodiments, amino acid 17 of SEQ ID NO:67 issubstituted with a phenylalanine or a leucine. In some embodiments,amino acid 23 of SEQ ID NO:67 is substituted with a proline. In someembodiments, amino acid 24 of SEQ ID NO:67 is substituted with anisoleucine or a phenylalanine. In some embodiments, amino acid 25 of SEQID NO:67 is substituted with a valine, an isoleucine, or an alanine. Insome embodiments, amino acid 26 of SEQ ID NO:67 is substituted with ahistidine, a tyrosine, or a histidine. In some embodiments, amino acid25 of SEQ ID NO:67 is substituted with a valine. In some embodiments,amino acid 26 of SEQ ID NO:67 is substituted with a leucine. In someembodiments, the signal sequence of the polypeptide comprises orconsists of a sequence selected from the group listed in Table 1.

TABLE 1 MEWGYLLEVTSLLAALALLQRSSGAAA SEQ ID NO: 67MEWGYLLEVTSLLAALALLQRSSGALA SEQ ID NO: 68 MEWGYLLEVTSLLAALALLQRSSGVLASEQ ID NO: 69 MEWGYLLEVTSLLAALLLLQRSPIVHA SEQ ID NO: 70MEWGYLLEVTSLLAALFLLQRSPIVHA SEQ ID NO: 71 MEWGYLLEVTSLLAALLLLQRSPFVHASEQ ID NO: 72 MEWGYLLEVTSLLAALLLLQRSPIIYA SEQ ID NO: 73MEWGYLLEVTSLLAALLLLQRSPIAHA SEQ ID NO: 74

In certain embodiments, the Wnt-binding agent comprises a firstpolypeptide comprising a FZD domain component and a Fc region. In someembodiments, the FZD domain component is from FZD 1, FZD2, FZD3, FZD4,FZD5, FZD6, FZD7, FZD8, FZD9, or FZD10. In some embodiments, the Fcregion is from an IgG1 immunoglobulin. In some embodiments, theWnt-binding agent comprises: (a) a first polypeptide consistingessentially of amino acids selected from the group consisting of: X1 toY1 of SEQ ID NO:27, X2 to Y2 of SEQ ID NO:28, X3 to Y3 of SEQ ID NO:29,X4 to Y4 of SEQ ID NO:22, X5 to Y5 of SEQ ID NO:23, X6 to Y6 of SEQ IDNO:24, X7 to Y7 of SEQ ID NO:25, X8 to Y8 of SEQ ID NO:30, X9 to Y9 ofSEQ ID NO:31, and X10 to Y10 of SEQ ID NO:26; and (b) a secondpolypeptide consisting essentially of amino acids A to B of SEQ IDNO:43;

wherein X1=amino acid 69, 70, 71,72, 73, 74, 75, or 76

Y1=amino acid 236, 237, 238, 239, 240, 241, 242, or 243

X2=amino acid 22, 23, 24, 25, 26, 27 or 28

Y2=amino acid 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171 or 172

X3=amino acid 18, 19, 20, 21, 22, 23, 24, or 25

Y3=amino acid 141, 142, 143, 144, 145, 146, 147, 148, or 149

X4=amino acid 38, 39, 40, 41, or 42

Y4=amino acid 168, 169, 170, 171, 172, 173, 174, 175 or 176

X5=amino acid 25, 26, 27, 28 or 29

Y5=amino acid 155, 156, 157, 158, 159, 160, 161, 162, 163, or 164

X6=amino acid 19, 20, 21, 22, 23, or 24

Y6=amino acid 144, 145, 146, 147, 148, 149, 150, 151 or 152

X7=amino acid 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34

Y7=amino acid 178, 179, 180, 181, 182, 183, 184, 185, or 186

X8=amino acid 25, 26, 27, 28, 29, 30, or 31

Y8=amino acid 156, 157, 158, 159, 160, 161, 162, 163, or 164

X9=amino acid 21, 22, 23, or 24

Y9=amino acid 137, 138, 139, 140, 141, 142, 143, 144, 145, or 146

X10=amino acid 20, 21, 22, 23, 24, or 25

Y10=amino acid 152, 153, 154, 155, 156, 157, 158, 159, or 160

A=amino acid 1, 2, 3, 4, 5, or 6

B=amino acid 231 or 232.

In some embodiments, the first polypeptide is directly linked to thesecond polypeptide. In some embodiments, the first polypeptide is linkedto the second polypeptide via a peptide linker In some embodiments, thefirst polypeptide is linked to the second polypeptide via the peptidelinker GRA. A polypeptide (e.g., a first or second polypeptide) that“consists essentially of” certain amino acids or is “consistingessentially of” certain amino acids may, in some embodiments, includeone or more (e.g., one, two, three, four or more) additional amino acidsat one or both ends, so long as the additional amino acids do notmaterially affect the function of the Wnt-binding agent.

In certain embodiments, the Wnt-binding agent comprises: (a) a firstpolypeptide consisting essentially of amino acids X to Y of SEQ IDNO:30; and (b) a second polypeptide consisting essentially of aminoacids A to B of SEQ ID NO:43; wherein the first polypeptide is directlylinked to the second polypeptide; and wherein

X=amino acid 25, 26, 27, 28, 29, 30, or 31

-   -   Y=amino acid 156, 157, 158, 159, 160, 161, 162, 163, or 164    -   A=amino acid 1, 2, 3, 4, 5, or 6    -   B=amino acid 231 or 232.        In some embodiments, the first polypeptide consists essentially        of amino acids 25-158 of SEQ ID NO:30. In other embodiments, the        first polypeptide consists of amino acids 25-158 of SEQ ID        NO:30. In some embodiments, the first polypeptide consists        essentially of amino acids 28-158 of SEQ ID NO:30. In other        embodiments, the first polypeptide consists of amino acids        28-158 of SEQ ID NO:30. In some embodiments, the first        polypeptide consists of amino acids 31-158 of SEQ ID NO:30. In        some embodiments, the second polypeptide consists of amino acids        1-232 of SEQ ID NO:43. In some embodiments, the second        polypeptide consists of amino acids 3-232 of SEQ ID NO:43. In        some embodiments, the second polypeptide consists of amino acids        6-232 of SEQ ID NO:43. In some embodiments, the first        polypeptide is SEQ ID NO:39 and the second polypeptide is SEQ ID        NO:43. In some embodiments, the first polypeptide is SEQ ID        NO:39 and the second polypeptide is SEQ ID NO:42. In some        embodiments, the first polypeptide is SEQ ID NO:39 and the        second polypeptide is SEQ ID NO:18.

In some embodiments, the Wnt-binding agent is a polypeptide comprising afirst polypeptide and a second polypeptide, wherein the polypeptides areselected from Table 2.

TABLE 2 First Polypeptide Second Polypeptide Amino acids 25-158 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-158 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-158 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-158 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-158 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-161 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-161 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-161 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-161 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 25-164 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 26-164 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 27-164 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 1-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 1-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 2-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 2-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 3-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 3-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 4-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 4-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 5-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 5-231 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 6-232 of SEQ ID NO: 30 SEQ ID NO: 43 Amino acids 28-164 of Aminoacids 6-231 of SEQ ID NO: 30 SEQ ID NO: 43

In some embodiments, the Wnt-binding agent comprises an amino acidsequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ IDNO:65, and SEQ ID NO:66.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:1. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:1, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:1. In certain embodiments, the variants of SEQ ID NO:1maintain the ability to bind one or more human Wnts.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:46. In some embodiments, the Wnt-binding agent is SEQ IDNO:46. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:46, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:46. In certain embodiments, the variants of SEQ ID NO:46maintain the ability to bind one or more human Wnts.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:48. In some embodiments, the Wnt-binding agent is SEQ IDNO:48. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:48, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:48. In certain embodiments, the variants of SEQ ID NO:48maintain the ability to bind one or more human Wnts.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:50. In some embodiments, the Wnt-binding agent is SEQ IDNO:50. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:50, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:50. In certain embodiments, the variants of SEQ ID NO:50maintain the ability to bind one or more human Wnts.

In certain embodiments, the Wnt-binding agent comprises the sequence ofSEQ ID NO:53. In some embodiments, the Wnt-binding agent is SEQ IDNO:53. In certain alternative embodiments, the agent comprises thesequence of SEQ ID NO:53, comprising one or more (e.g., one, two, three,four, five, six, seven, eight, nine, ten, etc.) conservativesubstitutions. In certain embodiments, the agent comprises a sequencehaving at least about 90%, about 95%, or about 98% sequence identitywith SEQ ID NO:53. In certain embodiments, the variants of SEQ ID NO:53maintain the ability to bind one or more human Wnts.

In some embodiments, the Wnt-binding agents as described herein inhibitthe growth of a tumor or tumor cells. In some embodiments, theWnt-binding agents induce cells in a tumor to differentiate. In someembodiments, the Wnt-binding agents induce the expression ofdifferentiation markers on a tumor or tumor cell. In certainembodiments, the Wnt-binding agents reduce the frequency of cancer stemcells in a tumor. In some embodiments, a Wnt-binding agent comprisingSEQ ID NO:46 inhibits tumor growth to a greater extent than aWnt-binding agent comprising SEQ ID NO:1. In some embodiments, aWnt-binding agent comprising SEQ ID NO:48 inhibits tumor growth to agreater extent than a Wnt-binding agent comprising SEQ ID NO:1. In someembodiments, a Wnt-binding agent comprising SEQ ID NO:50 inhibits tumorgrowth to a greater extent than a Wnt-binding agent comprising SEQ IDNO:1. In some embodiments, a Wnt-binding agent comprising SEQ ID NO:53inhibits tumor growth to a greater extent than a Wnt-binding agentcomprising SEQ ID NO:1. In some embodiments, a Wnt-binding agent asdescribed herein inhibits tumor growth to a greater extent than aWnt-binding agent comprising a FZD domain component, a Fc domain and alinker component connecting the FZD domain component and the Fc domain.In some embodiments, the linker component is an intervening peptidelinker

In certain embodiments, the Wnt-binding agents as described hereininhibit the growth of a Wnt-dependent tumor. In some embodiments, thetumor is a tumor selected from selected from the group consisting ofcolorectal tumor, colon tumor, pancreatic tumor, lung tumor, ovariantumor, liver tumor, breast tumor, kidney tumor, prostate tumor,gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,glioblastoma, and head and neck tumor. In certain embodiments, the tumoris a colorectal tumor. In certain embodiments, the tumor is a pancreatictumor. In certain embodiments, the tumor is a breast tumor.

In certain embodiments, a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:45, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:65 and SEQ IDNO:66 is provided. In certain embodiments, the polypeptide comprises anamino acid sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, and SEQ ID NO:53. In someembodiments, a polypeptide consists of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:46, SEQ ID NO:48,SEQ ID NO:50, and SEQ ID NO:53. In certain embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO:50. In some embodiments,the polypeptide is SEQ ID NO:50. In certain embodiments, the polypeptidecomprises the amino acid sequence of SEQ ID NO:53. In some embodiments,the polypeptide is SEQ ID NO:53.

In certain embodiments, the polypeptide (before signal sequencecleavage) comprises SEQ ID NO:50 and a signal sequence selected from thegroup consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74. Incertain embodiments, the polypeptide (before signal sequence cleavage)comprises SEQ ID NO:50 and a signal sequence selected from the groupconsisting of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73,and SEQ ID NO:74. In some embodiments, the polypeptide (before signalsequence cleavage) comprises SEQ ID NO:53 and a signal sequence selectedfrom the group consisting of SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ IDNO:74. In some embodiments, the polypeptide (before signal sequencecleavage) comprises SEQ ID NO:53 and a signal sequence selected from thegroup consisting of SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, and SEQ ID NO:74. In some embodiments, the polypeptide comprisesSEQ ID NO:71 and SEQ ID NO:50. In some embodiments, the polypeptidecomprises SEQ ID NO:71 and SEQ ID NO:53. In some embodiments, thepolypeptide comprises SEQ ID NO:75. In some embodiments, the polypeptideconsists essentially of SEQ ID NO:75.

In some embodiments, the polypeptide is a substantially purifiedpolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, andSEQ ID NO:53. In certain embodiments, the substantially purifiedpolypeptide consists of at least 90% of a polypeptide that has anN-terminal sequence of ASA. In some embodiments, the nascent polypeptidecomprises a signal sequence selected from the group consisting of SEQ IDNOs:67-74. In some embodiments, the nascent polypeptide comprises asignal sequence of SEQ ID NOs:71. In some embodiments, the nascentpolypeptide comprises a signal sequence that results in a substantiallyhomogeneous polypeptide product with one N-terminal sequence.

In certain alternative embodiments, the agent does not comprise a Fridomain of a FZD receptor.

In certain embodiments, the Wnt-binding agent is an antibody (e.g., anantibody that specifically binds to one or more Wnt proteins).

In certain embodiments, the Wnt-binding agent comprises a Fc region ofan immunoglobulin. Those skilled in the art will appreciate that thebinding agents of this invention will comprise fusion proteins in whichat least a portion of the Fc region has been deleted or otherwisealtered so as to provide desired biochemical characteristics, such asincreased cancer cell localization, increased tumor penetration, reducedserum half-life, or increased serum half-life, when compared with afusion protein of approximately the same immunogenicity comprising anative or unaltered constant region. Modifications to the Fc region mayinclude additions, deletions, or substitutions of one or more aminoacids in one or more domains. The modified fusion proteins disclosedherein may comprise alterations or modifications to one or more of thetwo heavy chain constant domains (CH2 or CH3) or to the hinge region. Inother embodiments, the entire CH2 domain is removed (ΔCH2 constructs).In some embodiments, the omitted constant region domain is replaced by ashort amino acid spacer (e.g., 10 aa residues) that provides some of themolecular flexibility typically imparted by the absent constant regiondomain.

In some embodiments, the modified fusion proteins are engineered to linkthe CH3 domain directly to the hinge region of the antibody. In otherembodiments, a peptide spacer is inserted between the hinge region andthe modified CH2 and/or CH3 domains. For example, constructs may beexpressed wherein the CH2 domain has been deleted and the remaining CH3domain (modified or unmodified) is joined to the hinge region with a5-20 amino acid spacer. Such a spacer may be added to ensure that theregulatory elements of the constant domain remain free and accessible orthat the hinge region remains flexible. However, it should be noted thatamino acid spacers may, in some cases, prove to be immunogenic andelicit an unwanted immune response against the construct. Accordingly,in certain embodiments, any spacer added to the construct will berelatively non-immunogenic so as to maintain the desired biologicalqualities of the modified antibodies.

In some embodiments, the modified fusion proteins may have only apartial deletion of a constant domain or substitution of a few or even asingle amino acid. For example, the mutation of a single amino acid inselected areas of the CH2 domain may be enough to substantially reduceFc binding and thereby increase cancer cell localization and/or tumorpenetration. Similarly, it may be desirable to simply delete that partof one or more constant region domains that control a specific effectorfunction (e.g., complement C1q binding) to be modulated. Such partialdeletions of the constant regions may improve selected characteristicsof the antibody (e.g., serum half-life) while leaving other desirablefunctions associated with the subject constant region domain intact.Moreover, as alluded to above, the constant regions of the disclosedfusion proteins may be modified through the mutation or substitution ofone or more amino acids that enhances the profile of the resultingconstruct. In this respect it may be possible to disrupt the activityprovided by a conserved binding site (e.g., Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. In certain embodiments, the modified fusionproteins comprise the addition of one or more amino acids to theconstant region to enhance desirable characteristics such as decreasingor increasing effector function, or provide for more cytotoxin orcarbohydrate attachment.

It is known in the art that the constant region mediates severaleffector functions. For example, binding of the C1 component ofcomplement to the Fc region of IgG or IgM antibodies (bound to antigen)activates the complement system. Activation of complement is importantin the opsonization and lysis of cell pathogens. The activation ofcomplement also stimulates the inflammatory response and can also beinvolved in autoimmune hypersensitivity. In addition, the Fc region ofan antibody can bind to a cell expressing a Fc receptor (FcR). There area number of Fc receptors which are specific for different classes ofantibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA(alpha receptors) and IgM (mu receptors). Binding of antibody to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction ofantibody-coated particles, clearance of immune complexes, lysis ofantibody-coated target cells by killer cells (antibody-dependentcell-mediated cytotoxicity or ADCC), release of inflammatory mediators,placental transfer, and control of immunoglobulin production.

In some embodiments, the Wnt-binding agents provide for altered effectorfunctions that, in turn, affect the biological profile of theadministered agent. For example, in some embodiments, the deletion orinactivation (through point mutations or other means) of a constantregion domain may reduce Fc receptor binding of the circulating modifiedagent (e.g., Wnt-binding agent) thereby increasing cancer celllocalization and/or tumor penetration. In other embodiments, theconstant region modifications increase or reduce the serum half-life ofthe agent. In some embodiments, the constant region is modified toeliminate disulfide linkages or oligosaccharide moieties.

In certain embodiments, a Wnt-binding agent does not have one or moreeffector functions normally associated with an Fc region. In someembodiments, the agent has no ADCC activity, and/or nocomplement-dependent cytotoxicity (CDC) activity. In certainembodiments, the agent does not bind to the Fc receptor and/orcomplement factors. In certain embodiments, the agent has no effectorfunction.

In some embodiments, the Wnt-binding agents described herein aremodified to reduce immunogenicity. In general, immune responses againstcompletely normal human proteins are rare when these proteins are usedas therapeutics. However, although many fusion proteins comprisepolypeptides sequences that are the same as the sequences found innature, several therapeutic fusion proteins have been shown to beimmunogenic in mammals. In some studies, a fusion protein comprising alinker has been found to be more immunogenic than a fusion protein thatdoes not contain a linker Accordingly, in some embodiments, thepolypeptides of the invention are analyzed by computation methods topredict immunogenicity. In some embodiments, the polypeptides areanalyzed for the presence of T-cell and/or B-cell epitopes. If anyT-cell or B-cell epitopes are identified and/or predicted, modificationsto these regions (e.g., amino acid substitutions) may be made to disruptor destroy the epitopes. Various algorithms and software that can beused to predict T-cell and/or B-cell epitopes are known in the art. Forexample, the software programs SYFPEITHI, HLA Bind, PEPVAC, RANKPEP,DiscoTope, ElliPro and Antibody Epitope Prediction are all publiclyavailable.

In some embodiments, a cell producing any of the Wnt-binding agents orpolypeptides described herein is provided. In some embodiments, acomposition comprising any of the Wnt-binding agents or polypeptidesdescribed herein is provided. In some embodiments, the compositioncomprises a polypeptide wherein at least 80%, 90%, 95%, 97%, 98%, or 99%of the polypeptide has an N-terminal sequence of ASA. In someembodiments, the composition comprises a polypeptide wherein 100% of thepolypeptide has an N-terminal sequence of ASA. In some embodiments, thecomposition comprises a polypeptide wherein at least 80% of thepolypeptide has an N-terminal sequence of ASA. In some embodiments, thecomposition comprises a polypeptide wherein at least 90% of thepolypeptide has an N-terminal sequence of ASA. In some embodiments, thecomposition comprises a polypeptide wherein at least 95% of thepolypeptide has an N-terminal sequence of ASA.

The polypeptides of the present invention can be recombinantpolypeptides, natural polypeptides, or synthetic polypeptides. It willbe recognized in the art that some amino acid sequences of the inventioncan be varied without significant effect on the structure or function ofthe protein. If such differences in sequence are contemplated, it shouldbe remembered that there will be critical areas on the protein whichdetermine activity. Thus, the invention further includes variations ofthe polypeptides which show substantial activity or which includeregions of FZD proteins, SFRP proteins or Ror proteins such as theprotein portions discussed herein. Such mutants include deletions,insertions, inversions, repeats, and type substitutions. As indicatedbelow, guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, et al., 1990, Science,247:1306-10.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above. Incertain embodiments, the number of substitutions for any given solublereceptor polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5or 3.

Fragments or portions of the polypeptides of the present invention canbe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments can be employed asintermediates for producing the full-length polypeptides. Thesefragments or portion of the polypeptides can also be referred to as“protein fragments” or “polypeptide fragments”.

A protein fragment of this invention is a portion, or all, of a proteinwhich is capable of binding to one or more human Wnt proteins (e.g., ahuman FZD receptor, a human SFRP or a Ror protein). In some embodiments,the fragment has a high affinity for one or more human Wnt proteins.Some fragments of fusion proteins described herein are protein fragmentscomprising at least part of the extracellular portion of a FZD receptor,a SFRP or the extracellular portion of a Ror protein which contains abinding domain linked to at least part of a constant region of animmunoglobulin (e.g., a Fc region). The binding affinity of the proteinfragment can be in the range of about 10⁻¹¹ to 10⁻¹² M, although theaffinity can vary considerably with fragments of different sizes,ranging from 10⁻⁷ to 10⁻¹³ M. In some embodiments, the fragment is about100 to about 200 amino acids in length and comprises a binding domainlinked to at least part of a constant region of an immunoglobulin.

The Wnt-binding agents of the present invention can be assayed forspecific binding by any method known in the art. The immunoassays whichcan be used include, but are not limited to, competitive andnon-competitive assay systems using techniques such as BIAcore analysis,FACS analysis, immunofluorescence, immunocytochemistry, Western blots,radioimmunoassays, ELISA, “sandwich” immunoassays, immunoprecipitationassays, precipitation reactions, gel diffusion precipitin reactions,immunodiffusion assays, agglutination assays, complement-fixationassays, immunoradiometric assays, fluorescent immunoassays, and proteinA immunoassays. Such assays are routine and well known in the art (see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated byreference herein in its entirety).

For example, the specific binding of a polypeptide to a human Wnt may bedetermined using ELISA. An ELISA assay comprises preparing antigen,coating wells of a 96 well microtiter plate with antigen, adding thepolypeptide (e.g., a Wnt-binding agent) conjugated to a detectablecompound such as an enzymatic substrate (e.g. horseradish peroxidase oralkaline phosphatase) to the well, incubating for a period of time anddetecting the presence of the agent. In some embodiments, thepolypeptide (e.g., Wnt-binding agent) is not conjugated to a detectablecompound, but instead a second conjugated antibody that recognizes thepolypeptide is added to the well. In some embodiments, instead ofcoating the well with the antigen, the polypeptide (e.g., Wnt-bindingagent) can be coated to the well and a second antibody conjugated to adetectable compound can be added following the addition of the antigento the coated well. One of skill in the art would be knowledgeable as tothe parameters that can be modified to increase the signal detected aswell as other variations of ELISAs known in the art (see e.g. Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 11.2.1).

The binding affinity of an agent to a Wnt and the off-rate of a bindingagent-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I), orfragment or variant thereof, with the binding agent of interest in thepresence of increasing amounts of unlabeled antigen followed by thedetection of the antibody bound to the labeled antigen. The affinity ofthe binding agent against a Wnt and the binding off-rates can bedetermined from the data by Scatchard plot analysis. In someembodiments, BIAcore kinetic analysis is used to determine the bindingon and off rates of agents that bind one or more human Wnts. BIAcorekinetic analysis comprises analyzing the binding and dissociation ofantibodies from chips with immobilized Wnt antigens on their surface.

In certain embodiments, the Wnt-binding agent binds to at least one Wntwith a dissociation constant (K_(D)) of about 1 μM or less, about 100 nMor less, about 40 nM or less, about 20 nM or less, or about 10 nM orless.

In certain embodiments, the Wnt-binding agent (e.g., a FZD8-Fc) is anantagonist of at least one Wnt (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10Wnts) bound by the agent. In certain embodiments, the agent inhibits atleast about 10%, at least about 20%, at least about 30%, at least about50%, at least about 75%, at least about 90%, or about 100% of one ormore activity of the bound human Wnt(s).

In vivo and in vitro assays for determining whether a Wnt-binding agent(or candidate Wnt-binding agent) inhibits Wnt signaling are known in theart. For example, cell-based, luciferase reporter assays utilizing aTCF/Luc reporter vector containing multiple copies of the TCF-bindingdomain upstream of a firefly luciferase reporter gene may be used tomeasure canonical Wnt signaling levels in vitro (Gazit et al., 1999,Oncogene 18; 5959-66). The level of Wnt signaling in the presence of oneor more Wnts (e.g., Wnt(s) expressed by transfected cells or provided byWnt-conditioned media) with the Wnt-binding agent present is compared tothe level of signaling without the Wnt-binding agent present. Inaddition to the TCF/Luc reporter assay, the effect of a Wnt-bindingagent (or candidate agent) on canonical Wnt signaling may be measured invitro or in vivo by measuring the effect of the agent on the level ofexpression of β-catenin regulated genes, such as c-myc (He et al.,Science, 281:1509-12 (1998)), cyclin D1 (Tetsu et al., Nature, 398:422-6(1999)) and/or fibronectin (Gradl et al. Mol. Cell Biol., 19:5576-87(1999)). In certain embodiments, the effect of an agent on Wnt signalingmay also be assessed by measuring the effect of the agent on thephosphorylation state of Dishevelled-1, Dishevelled-2, Dishevelled-3,LRP5, LRP6, and/or β-catenin.

The polypeptides described herein can be produced by any suitable methodknown in the art. Such methods range from direct protein synthesismethods to constructing DNA sequences that encode polypeptide sequencesand expressing those sequences in a suitable host. In some embodiments,a DNA sequence is constructed using recombinant technology by isolatingor synthesizing a DNA sequence encoding a wild-type protein of interest.Optionally, the sequence can be mutagenized by site-specific mutagenesisto provide functional analogs thereof. See, e.g., Zoeller et al., 1984,PNAS, 81:5662-66 and U.S. Pat. No. 4,588,585. In some embodiments, a DNAsequence is constructed using recombinant technology by isolating morethan one DNA sequence encoding two polypeptides of interest and ligatingthese DNA sequences together to generate a fusion protein. In someembodiments, the fusion of the two polypeptides adds additional aminoacids to the junction between the two polypeptides (i.e., the ligationsite for the DNA sequences). These additional amino acids are considereda linker In some embodiments, a peptide linker is inserted between thetwo polypeptides of the fusion protein.

In some embodiments, a DNA sequence that encodes a polypeptide ofinterest may be constructed by chemical synthesis using anoligonucleotide synthesizer. Such oligonucleotides can be designed basedon the amino acid sequence of the desired polypeptide. In someembodiments, the oligonucleotides are designed to select codons that arefavored in the host cell in which the recombinant polypeptide ofinterest will be produced. Standard methods can be applied to synthesizea polynucleotide sequence that encodes a polypeptide of interest. Forexample, a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular polypeptide can be synthesized. Forexample, several small oligonucleotides coding for portions of thedesired polypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly. In some embodiments, a nucleotide sequence coding for thedesired fusion protein is synthesized so that the two polypeptides aredirectly linked without an intervening peptide linker

Once assembled (by synthesis, site-directed mutagenesis, recombinanttechnology, or another method), the polynucleotide sequences encoding aparticular polypeptide of interest can be inserted into an expressionvector and operatively linked to an expression control sequenceappropriate for expression of the polypeptide in a desired host. Properassembly can be confirmed by nucleotide sequencing, restriction mapping,and/or expression of a biologically active polypeptide in a suitablehost. As is well-known in the art, in order to obtain high expressionlevels of a transfected gene in a host, the gene must be operativelylinked to transcriptional and translational expression control sequencesthat are functional in the chosen expression host.

In certain embodiments, recombinant expression vectors are used toamplify and express DNA that encode Wnt-binding agents and polypeptidesdescribed herein. For example, recombinant expression vectors can bereplicable DNA constructs which have synthetic or cDNA-derived DNAfragments encoding a fusion protein comprising a FZD Fri domain and a Fcregion, operatively linked to suitable transcriptional or translationalregulatory elements derived from mammalian, microbial, viral or insectgenes. A transcriptional unit generally comprises an assembly of (1) agenetic element or elements having a regulatory role in gene expression,for example, transcriptional promoters and/or enhancers, (2) astructural or coding sequence which is transcribed into mRNA andtranslated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences. Regulatory elementscan include an operator sequence to control transcription. The abilityto replicate in a host, usually conferred by an origin of replication,and a selection gene to facilitate recognition of transformants canadditionally be incorporated. DNA regions are “operatively linked” whenthey are functionally related to each other. For example, DNA for asignal peptide (secretory leader) is operatively linked to DNA for apolypeptide if it is expressed as a precursor which participates in thesecretion of the polypeptide; a promoter is operatively linked to acoding sequence if it controls the transcription of the sequence; or aribosome binding site is operatively linked to a coding sequence if itis positioned so as to permit translation. Generally, operatively linkedmeans contiguous and, in the case of secretory leaders, means contiguousand in reading frame. In some embodiments, structural elements intendedfor use in yeast expression systems can include a leader sequenceenabling extracellular secretion of translated protein by a host cell.In some embodiments, where recombinant protein is expressed without aleader or transport sequence, it can include an N-terminal methionineresidue. This residue can optionally be subsequently cleaved from theexpressed recombinant protein to provide a final product.

The choice of an expression control sequence and expression vectordepends upon the choice of host. A wide variety of expressionhost/vector combinations can be employed. Useful expression vectors foreukaryotic hosts include, for example, vectors comprising expressioncontrol sequences from SV40, bovine papilloma virus, adenovirus andcytomegalovirus. Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, including pCR1,pBR322, pMB9 and their derivatives, and wider host range vectors, suchas M13 and other filamentous single-stranded DNA phages.

Suitable host cells for expression of a Wnt-binding agent includeprokaryotes, yeast, insect, or higher eukaryotic cells under the controlof appropriate promoters. Prokaryotes include gram-negative orgram-positive organisms, for example E. coli or Bacilli. Highereukaryotic cells include established cell lines of mammalian origin asdescribed below. Cell-free translation systems can also be employed.Appropriate cloning and expression vectors for use with bacterial,fungal, yeast, and mammalian cellular hosts are described by Pouwels etal., 1985, Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., therelevant disclosure of which is hereby incorporated by reference.

Various mammalian or insect cell culture systems are used to expressrecombinant polypeptides. In some embodiments, expression of recombinantproteins in mammalian cells is preferred because such proteins aregenerally correctly folded, appropriately modified and completelyfunctional. Examples of suitable mammalian host cell lines include theCOS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127(murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO(Chinese hamster ovary-derived), HeLa (human cervical cancer-derived)and BHK (hamster kidney fibroblast-derived) cell lines. Mammalianexpression vectors can comprise non-transcribed elements such as anorigin of replication, a suitable promoter and enhancer linked to thegene to be expressed, and other 5′ or 3′ flanking non-transcribedsequences, and 5′ or 3′ non-translated sequences, such as necessaryribosome binding sites, a polyadenylation site, splice donor andacceptor sites, and transcriptional termination sequences. Baculovirussystems for production of heterologous proteins in insect cells areknown to those of skill in the art and are reviewed by Luckow andSummers, 1988, Bio/Technology, 6:47.

The proteins produced by a transformed host can be purified according toany suitable method. Such standard methods include chromatography (e.g.,ion exchange, affinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for protein purification. Affinity tags such as hexahistidine,maltose binding domain, influenza coat sequence, andglutathione-S-transferase can be attached to the protein to allow easypurification by passage over an appropriate affinity column. Isolatedproteins can also be physically characterized using such techniques asproteolysis, mass spectrometry (MS), high performance liquidchromatography (HPLC), nuclear magnetic resonance (NMR), and x-raycrystallography.

In some embodiments, supernatants from expression systems which secreterecombinant protein into culture media can be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix. In some embodiments, an anion exchange resin can beemployed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose, or other types commonly employed in proteinpurification. In some embodiments, a cation exchange step can beemployed. Suitable cation exchangers include various insoluble matricescomprising sulfopropyl or carboxymethyl groups. In some embodiments, ahydroxyapatite (CHT) media can be employed, including but not limitedto, ceramic hydroxyapatite. In some embodiments, one or morereversed-phase HPLC steps employing hydrophobic RP-HPLC media, e.g.,silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify a fusion protein. Some or all of theforegoing purification steps, in various combinations, can also beemployed to provide a homogeneous recombinant protein.

In some embodiments, recombinant protein produced in bacterial culturecan be isolated, for example, by initial extraction from cell pellets,followed by one or more concentration, salting-out, aqueous ionexchange, and/or size exclusion chromatography steps. HPLC can beemployed for final purification steps. Microbial cells employed inexpression of a recombinant protein can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Methods known in the art for purifying antibodies and other proteinsalso include, for example, those described in U.S. Patent Appl. Nos.2008/0312425; 2008/0177048; and 2009/0187005.

The polypeptides described herein can be further modified to containadditional chemical moieties not normally part of the protein. Thosederivatized moieties can improve the solubility, the biological halflife, or absorption of the protein. The moieties can also reduce oreliminate any undesirable side effects of the proteins and the like. Anoverview for those moieties can be found in Remington: The Science andPractice of Pharmacy, 21^(st) Edition, University of the Sciences,Philadelphia, 2005.

The chemical moieties most suitable for derivatization include watersoluble polymers. A water soluble polymer is desirable because theprotein to which it is attached does not precipitate in an aqueousenvironment, such as a physiological environment. In some embodiments,the polymer will be pharmaceutically acceptable for the preparation of atherapeutic product or composition. One skilled in the art will be ableto select the desired polymer based on such considerations as whetherthe polymer/protein conjugate will be used therapeutically, and if so,the desired dosage, circulation time, resistance to proteolysis, andother considerations. The effectiveness of the derivatization can beascertained by administering the derivative, in the desired form (i.e.,by osmotic pump, or by injection or infusion, or, further formulated fororal, pulmonary or other delivery routes), and determining itseffectiveness. Suitable water soluble polymers include, but are notlimited to, polyethylene glycol (PEG), copolymers of ethyleneglycol/propylene glycol, carboxymethylcellulose, dextran, polyvinylalcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymersor random copolymers), dextran, poly(n-vinyl pyrrolidone)-polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde canhave advantages in manufacturing due to its stability in water.

The number of polymer molecules so attached can vary, and one skilled inthe art will be able to ascertain the effect on function. One canmono-derivatize, or can provide for a di-, tri-, tetra- or somecombination of derivatization, with the same or different chemicalmoieties (e.g., polymers, such as different weights of polyethyleneglycols). The proportion of polymer molecules to protein (or peptide)molecules will vary, as will their concentrations in the reactionmixture. In general, the optimum ratio (in terms of efficiency ofreaction in that there is no excess unreacted protein or polymer) willbe determined by factors such as the desired degree of derivatization(e.g., mono-, di-, tri-, etc.), the molecular weight of the polymerselected, whether the polymer is branched or unbranched, and thereaction conditions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art. See for example, EP0401384, the disclosure of which is hereby incorporated by reference(coupling PEG to G-CSF), see also Malik et al., 1992, Exp. Hematol.,20:1028-35 (reporting pegylation of GM-CSF using tresyl chloride). Forexample, polyethylene glycol can be covalently bound through amino acidresidues via a reactive group, such as, a free amino or carboxyl group.Reactive groups are those to which an activated polyethylene glycolmolecule can be bound. The amino acid residues having a free amino groupcan include lysine residues and the N-terminal amino acid residue. Thosehaving a free carboxyl group can include aspartic acid residues,glutamic acid residues, and the C-terminal amino acid residue.Sulfhydryl groups can also be used as a reactive group for attaching thepolyethylene glycol molecule(s). For therapeutic purposes, attachment atan amino group, such as attachment at the N-terminus or lysine group canbe performed. Attachment at residues important for receptor bindingshould be avoided if receptor binding is desired.

One can specifically design an amino-terminal chemically modifiedprotein. Using polyethylene glycol as an illustration of the presentcompositions, one can select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)can be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective N-terminal chemicalmodification can be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved. For example, onecan selectively N-terminally pegylate the protein by performing thereaction at a pH which allows one to take advantage of the pKadifferences between the epsilon amino group of the lysine residues andthat of the alpha amino group of the N-terminal residue of the protein.By such selective derivatization, attachment of a water soluble polymerto a protein is controlled, e.g., the conjugation with the polymer takesplace predominantly at the N-terminus of the protein and no significantmodification of other reactive groups, such as the lysine side chainamino groups, occurs. Using reductive alkylation, the water solublepolymer can be of the type described above, and should have a singlereactive aldehyde for coupling to the protein. Polyethylene glycolpropionaldehyde, containing a single reactive aldehyde, can be used.

Pegylation can be carried out by any of the pegylation reactions knownin the art. See, e.g., Focus on Growth Factors, 1992, 3: 4-10; EP0154316, the disclosure of which is hereby incorporated by reference; EP0401384; and the other publications cited herein that relate topegylation. The pegylation can be carried out via an acylation reactionor an alkylation reaction with a reactive polyethylene glycol molecule(or an analogous reactive water soluble polymer).

Thus, it is contemplated that soluble receptor polypeptides to be usedin accordance with the present invention can include pegylated solublereceptor proteins or variants, wherein the PEG group(s) is (are)attached via acyl or alkyl groups. Such products can be mono-pegylatedor poly-pegylated. The PEG groups are generally attached to the proteinat the α or ε amino groups of amino acids, but it is also contemplatedthat the PEG groups could be attached to any amino group attached to theprotein, which is sufficiently reactive to become attached to a PEGgroup under suitable reaction conditions.

The polymer molecules used in both the acylation and alkylationapproaches can be selected from among water soluble polymers asdescribed above. The polymer selected should be modified to have asingle reactive group, such as an active ester for acylation or analdehyde for alkylation, so that the degree of polymerization can becontrolled as provided for in the present methods. An exemplary reactivePEG aldehyde is polyethylene glycol propionaldehyde, which is waterstable, or mono C1-C10 alkoxy or aryloxy derivatives thereof (see U.S.Pat. No. 5,252,714). The polymer can be branched or unbranched. For theacylation reactions, the polymer(s) selected should have a singlereactive ester group. For the present reductive alkylation, thepolymer(s) selected should have a single reactive aldehyde group.Generally, the water soluble polymer will not be selected from naturallyoccurring glycosyl residues since these are usually made moreconveniently by mammalian recombinant expression systems. The polymercan be of any molecular weight, and can be branched or unbranched. Onewater soluble polymer for use herein is polyethylene glycol. As usedherein, polyethylene glycol is meant to encompass any of the forms ofPEG that have been used to derivatize other proteins, such as mono(C1-C10) alkoxy- or aryloxy-polyethylene glycol.

Other reaction parameters, such as solvent, reaction times,temperatures, etc., and means of purification of products, can bedetermined case by case based on the published information relating toderivatization of proteins with water soluble polymers (see thepublications cited herein). In certain embodiments, the Wnt-bindingagent is a polypeptide that is not derived from a human FZD or SFRP. Avariety of methods for identifying and producing polypeptides that bindwith high affinity to a protein target are known in the art. See, e.g.,Skerra, 2007, Curr. Opin. Biotechnol., 18:295-304; Hosse et al., 2006,Protein Science, 15:14-27; Gill et al., 2006, Curr. Opin. Biotechnol.,17:653-58; Nygren, 2008, FEBS J., 275:2668-76; and Skerra, 2008, FEBSJ., 275:2677-83, each of which is incorporated by reference herein inits entirety. In certain embodiments, phage display technology has beenused to identify/produce the Wnt-binding polypeptide. In certainembodiments, the polypeptide comprises a protein scaffold of a typeselected from the group consisting of protein A, a lipocalin, afibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.

In some embodiments, the Wnt-binding agent is a non-protein molecule. Incertain embodiments, the agent is a small molecule. Combinatorialchemistry libraries and techniques useful in the identification ofnon-protein Wnt-binding agents are known to those skilled in the art.See, e.g., Kennedy et al., 2008, J. Comb. Chem., 10:345-54; Dolle et al,2007, J. Comb. Chem., 9:855-902; and Bhattacharyya, 2001, Curr. Med.Chem., 8:1383-404, each of which is incorporated by reference herein inits entirety. In certain further embodiments, the agent is acarbohydrate, a glycosaminoglycan, a glycoprotein, or a proteoglycan.

In certain embodiments, the agent is a nucleic acid aptamer. Aptamersare polynucleotide molecules that are selected (e.g., from random ormutagenized pools) on the basis of their ability to bind to anothermolecule. In some embodiments, the aptamer comprises a DNApolynucleotide. In certain alternative embodiments, the aptamercomprises an RNA polynucleotide. In certain embodiments, the aptamercomprises one or more modified nucleic acid residues. Methods ofgenerating and screening nucleic acid aptamers for binding to proteinsare well known in the art. See, e.g., U.S. Pat. Nos. 5,270,163;5,683,867; 5,763,595; 6,344,321; 7,368,236; 5,582,981; 5,756,291;5,840,867; 7,312,325; and 7,329,742, International Patent PublicationNos. WO 02/077262 and WO 03/070984, U.S. Patent Application PublicationNos. 2005/0239134; 2005/0124565; and 2008/0227735, each of which isincorporated by reference herein in its entirety.

In certain embodiments, the Wnt-binding agent has a circulatinghalf-life in mice, cynomolgus monkeys, or humans of at least about 5hours, at least about 10 hours, at least about 24 hours, at least about3 days, at least about 1 week, or at least about 2 weeks. In certainembodiments, the Wnt-binding agent is an IgG (e.g., IgG1 or IgG2)antibody that has a circulating half-life in mice, cynomolgus monkeys,or humans of at least about 10 hours, at least about 24 hours, at leastabout 3 days, at least about 1 week, or at least about 2 weeks. Methodsof increasing the half-life of agents such as polypeptides andantibodies are known in the art. For example, known methods ofincreasing the circulating half-life of IgG antibodies include theintroduction of mutations in the Fc region which increase thepH-dependent binding of the antibody to the neonatal Fc receptor (FcRn)at pH 6.0 (see, e.g., U.S. Pat. Pub. Nos. 2005/0276799, 2007/0148164,and 2007/0122403). Known methods of increasing the circulating half-lifeof antibody fragments lacking the Fc region include such techniques aspegylation.

In certain embodiments, the Wnt-binding agents and polypeptides asdescribed herein have a half-life of at least about 50 hours in a ratwhen administered via the tail vein at a dose ranging from about 2 mg/kgto about 10 mg/kg. In certain embodiments, the Wnt-binding agent orpolypeptide has a half-life of at least about 50 hours in a rat whenadministered via the tail vein at a dose of about 10 mg/kg. In certainembodiments, the Wnt-binding agent or polypeptide has a half-life of atleast about 100 hours in a rat when administered via the tail vein at adose ranging from about 2 mg/kg to about 10 mg/kg. In certainembodiments, the Wnt-binding agent or polypeptide has a half-life of atleast about 100 hours in a rat when administered via the tail vein at adose of about 10 mg/kg. In certain embodiments, the Wnt-binding agenthas a half-life of at least about 120 hours in a rat when administeredvia the tail vein at a dose ranging from about 2 mg/kg to about 10mg/kg. In certain embodiments, the Wnt-binding agent has a half-life ofat least about 150 hours in a rat when administered via the tail vein ata dose ranging from about 2 mg/kg to about 10 mg/kg.

In certain embodiments, the agent is a soluble FZD receptor thatcomprises a Fri domain of a human FZD receptor (or a fragment or variantof the Fri domain that binds one or more Wnts) and a human Fc region andhas a half-life in vivo (e.g., in a mouse or rat) that is longer than asoluble FZD receptor comprising the extracellular domain of the FZDreceptor and a human Fc region.

Cells producing the Wnt-binding agents or polypeptides described hereinare provided. In some embodiments, the cell produces a solubleWnt-binding agent which comprises a Fri domain of human FZD8, wherein atleast about 80% of the Wnt-binding agent has an N-terminal sequence ofASA. In some embodiments, the cell produces a soluble Wnt-binding agentwhich comprises a Fri domain of human FZD8, wherein at least about 85%,at least about 90%, at least about 95%, or at least about 98% of theWnt-binding agent has an N-terminal sequence of ASA. In someembodiments, the cell is a mammalian cell. In some embodiments, the cellproduces a Wnt-binding agent which comprises a human Fc region. In someembodiments, the cell produces a Wnt-binding agent which comprises anamino acid sequence selected from the group consisting of SEQ ID NO:53,SEQ ID NO:50, SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:1. In someembodiments, the cell produces a Wnt-binding agent which comprises anamino acid sequence of SEQ ID NO:53. In some embodiments, the cellproduces a Wnt-binding agent which comprises an amino acid sequence ofSEQ ID NO:50.

Wnt-binding agents produced by the cells described herein are provided.

Compositions comprising the Wnt-binding agents or polypeptides describedherein are also provided. In some embodiments, the composition comprisesa soluble Wnt-binding agent which comprises a Fri domain of human FZD8,wherein at least about 80% of the Wnt-binding agent has an N-terminalsequence of ASA. In some embodiments, the composition comprises asoluble Wnt-binding agent which comprises a Fri domain of human FZD8,wherein at least about 85%, at least about 90%, at least about 95%, orat least about 98% of the Wnt-binding agent has an N-terminal sequenceof ASA. In some embodiments, the composition comprises a Wnt-bindingagent which comprises a human Fc region. In some embodiments, thecomposition comprises a Wnt-binding agent which comprises an amino acidsequence selected from the group consisting of SEQ ID NO:53, SEQ IDNO:50, SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:1. In some embodiments,the composition comprises a Wnt-binding agent which comprises an aminoacid sequence of SEQ ID NO:53. In some embodiments, the compositioncomprises a Wnt-binding agent which comprises an amino acid sequence ofSEQ ID NO:50. In some embodiments, the compositions as described hereinfurther comprise a pharmaceutically acceptable carrier.

Methods of using the compositions comprising the Wnt-binding agents orpolypeptides described herein are also provided.

III. Polynucleotides

In certain embodiments, the invention encompasses polynucleotidescomprising polynucleotides that encode a polypeptide that specificallybinds a human Wnt protein or a fragment of such a polypeptide. Forexample, the invention provides a polynucleotide comprising a nucleicacid sequence that encodes a soluble FZD receptor or encodes a fragmentof such a soluble receptor. In some embodiments, the invention providesa polynucleotide comprising a nucleic acid sequence that encodes asoluble SFRP, a soluble Ror protein or encodes a fragment of such asoluble protein. In some embodiments, the polynucleotides comprisepolynucleotides that encode any of the Wnt-binding agents as describedherein. The polynucleotides of the invention can be in the form of RNAor in the form of DNA. DNA includes cDNA, genomic DNA, and syntheticDNA; and can be double-stranded or single-stranded, and ifsingle-stranded can be the coding strand or non-coding (anti-sense)strand.

In certain embodiments, the polynucleotides are isolated. In certainembodiments, the polynucleotides are substantially pure.

The invention provides a polynucleotide comprising a polynucleotide thatencodes a polypeptide comprising the sequence of SEQ ID NO:1, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:75. In some embodiments,the polynucleotide further comprises a polynucleotide that encodes apolypeptide signal sequence selected from the group consisting of SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, and SEQ ID NO:74. In some embodiments, thepolynucleotide further comprises a polynucleotide that encodes apolypeptide signal sequence selected from the group consisting of SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74. Insome embodiments, the polynucleotide comprises a polynucleotide thatencodes a polypeptide having the sequence of SEQ ID NO:71 and SEQ IDNO:50. In some embodiments, the polynucleotide comprises apolynucleotide that encodes a polypeptide having the sequence of SEQ IDNO:71 and SEQ ID NO:53. In some embodiments, the polynucleotidecomprises a polynucleotide that encodes a polypeptide having thesequence of SEQ ID NO:75. The invention further provides apolynucleotide comprising the sequence of SEQ ID NO:2.

The invention provides a polynucleotide comprising a polynucleotide thatencodes a polypeptide comprising: a signal sequence selected from thegroup consisting of SEQ ID NO:71, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:73, and SEQ ID NO:74; a Fri domain of human FZD8; and a human Fcregion. In some embodiments, the polynucleotide comprises apolynucleotide that encodes a polypeptide comprising a signal sequenceof SEQ ID NO:71; a Fri domain of human FZD8; and a human Fc region.

Also provided is a polynucleotide that comprises a polynucleotide thathybridizes to a polynucleotide having the sequence of SEQ ID NO:2 and/orto a polynucleotide that encodes a polypeptide having the sequence ofSEQ ID NO:1, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ IDNO:54, SEQ ID NO:55, SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:75. Incertain embodiments, the hybridization is under conditions of highstringency.

In certain embodiments, the polynucleotides comprise the coding sequencefor the mature polypeptide joined in the same reading frame to apolynucleotide which aids, for example, in expression and secretion of apolypeptide from a host cell (e.g. a leader sequence or signal sequencewhich functions as a secretory sequence for controlling transport of apolypeptide from the cell). The polypeptide having a leader sequence isa preprotein and can have the leader sequence cleaved by the host cellto form the mature form of the polypeptide. The polynucleotides can alsoencode for a proprotein which is the mature protein plus additional 5′amino acid residues. A mature protein having a prosequence is aproprotein and is an inactive form of the protein. Once the prosequenceis cleaved an active mature protein remains.

In certain embodiments, the polynucleotides comprise the coding sequencefor the mature polypeptide joined in the same reading frame to a markersequence that allows, for example, for purification of the encodedpolypeptide. For example, the marker sequence can be a hexahistidine tagsupplied by a pQE-9 vector to provide for purification of the maturepolypeptide joined to the marker in the case of a bacterial host, or themarker sequence can be a hemagglutinin (HA) tag derived from theinfluenza hemagglutinin protein when a mammalian host (e.g., COS-7cells) is used.

The present invention further relates to variants of the hereinabovedescribed polynucleotides encoding, for example, fragments, analogs, andderivatives. Fragments or portions of the polynucleotides of the presentinvention can be used to synthesize full-length polynucleotides of thepresent invention.

In certain embodiments, the present invention provides isolatedpolynucleotides comprising polynucleotides having a nucleotide sequenceat least 80% identical, at least 85% identical, at least 90% identical,at least 95% identical, and in some embodiments, at least 96%, 97%, 98%or 99% identical to a polynucleotide encoding a polypeptide comprising asoluble FZD receptor or other Wnt-binding agent described herein.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence can include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence can be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence can be inserted intothe reference sequence. These mutations of the reference sequence canoccur at the amino- or carboxy-terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among nucleotides in the referencesequence or in one or more contiguous groups within the referencesequence.

The polynucleotide variants can contain alterations in the codingregions, non-coding regions, or both. In some embodiments thepolynucleotide variants contain alterations which produce silent aminoacid substitutions, additions, or deletions, but do not alter theproperties or activities of the encoded polypeptide. In someembodiments, nucleotide variants are produced by silent substitutionsdue to the degeneracy of the genetic code. In some embodiments,nucleotide variants comprise nucleotide sequences which result inexpression differences (e.g., increased or decreased expression), eventhough the amino acid sequence is not changed. Polynucleotide variantscan be produced for a variety of reasons, e.g., to optimize codonexpression for a particular host (change codons in the human mRNA tothose preferred by a bacterial host such as E. coli).

The polynucleotides described herein can be produced by any suitablemethod known in the art. As described herein in some embodiments, a DNAsequence is constructed using recombinant technology by isolating orsynthesizing a DNA sequence encoding a wild-type protein of interest. Insome embodiments, a DNA sequence is constructed using recombinanttechnology by isolating more than one DNA sequence encoding twopolypeptides of interest and ligating these DNA sequences together togenerate a fusion protein.

In some embodiments, a DNA sequence may be constructed by chemicalsynthesis using an oligonucleotide synthesizer. Such oligonucleotidescan be designed based on the amino acid sequence of the desiredpolypeptide. Standard methods can be applied to synthesize apolynucleotide sequence encoding a polypeptide of interest. For example,a complete amino acid sequence can be used to construct aback-translated gene. Further, a DNA oligomer containing a nucleotidesequence coding for the particular polypeptide can be synthesized. Forexample, several small oligonucleotides coding for portions of thedesired polypeptide can be synthesized and then ligated. The individualoligonucleotides typically contain 5′ or 3′ overhangs for complementaryassembly. In some embodiments, a nucleotide sequence coding for thedesired fusion protein is synthesized so that the two polypeptides aredirectly linked without an intervening peptide linker

Once assembled (by synthesis, site-directed mutagenesis, recombinanttechnology, or another method), the polynucleotide sequences encoding aparticular polypeptide of interest can be inserted into an expressionvector and operatively linked to an expression control sequenceappropriate for expression of the polypeptide in a desired host. Properassembly can be confirmed by nucleotide sequencing, restriction mapping,and/or expression of a biologically active polypeptide in a suitablehost. As is well-known in the art, in order to obtain high expressionlevels of a transfected gene in a host, the gene must be operativelylinked to transcriptional and translational expression control sequencesthat are functional in the chosen expression host.

Vectors comprising the polynucleotides described herein are provided.Cells comprising the vectors or polynucleotides described herein arealso provided.

IV. Pharmaceutical Compositions

The present invention further provides pharmaceutical compositionscomprising agents (e.g., soluble FZD receptors) that bind to one or moreWnt proteins and/or are Wnt antagonists. In some embodiments, thepharmaceutical compositions comprise the Wnt-binding agents andpolypeptides as described herein. These pharmaceutical compositions finduse in inhibiting tumor cell growth and treating cancer in humanpatients. In some embodiments, the Wnt-binding agents as describedherein find use in the manufacture of a medicament for the treatment ofcancer.

Formulations are prepared for storage and use by combining a purifiedagent or antagonist of the present invention with a pharmaceuticallyacceptable carrier, excipient, and/or stabilizer as a sterilelyophilized powder, aqueous solution, etc. (Remington: The Science andPractice of Pharmacy, 21^(st) Edition, University of the Sciences,Philadelphia, 2005). Suitable carriers, excipients, or stabilizerscomprise nontoxic buffers such as phosphate, citrate, and other organicacids; salts such as sodium chloride; antioxidants including ascorbicacid and methionine; preservatives (e.g. octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride;benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens,such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol;3-pentanol; and m-cresol); low molecular weight polypeptides (such asless than about 10 amino acid residues); proteins such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; carbohydrates such asmonosacchandes, disaccharides, glucose, mannose, or dextrins; chelatingagents such as EDTA; sugars such as sucrose, mannitol, trehalose orsorbitol; salt-forming counter-ions such as sodium; metal complexes(e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEENor polyethylene glycol (PEG).

The pharmaceutical compositions of the present invention can beadministered in any number of ways for either local or systemictreatment. Administration can be topical (such as to mucous membranesincluding vaginal and rectal delivery) such as transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders; pulmonary (e.g., by inhalation or insufflation of powdersor aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal); oral; or parenteral including intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial (e.g., intrathecal or intraventricular)administration.

The therapeutic formulation can be in unit dosage form. Suchformulations include tablets, pills, capsules, powders, granules,solutions or suspensions in water or non-aqueous media, or suppositoriesfor oral, parenteral, or rectal administration or for administration byinhalation. In solid compositions such as tablets the principal activeingredient is mixed with a pharmaceutical carrier. Conventionaltableting ingredients include corn starch, lactose, sucrose, sorbitol,talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, andother diluents (e.g. water) to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention,or a non-toxic pharmaceutically acceptable salt thereof. The solidpreformulation composition is then subdivided into unit dosage forms ofthe type described above. The tablets, pills, etc., of the novelcomposition can be coated or otherwise compounded to provide a dosageform affording the advantage of prolonged action. For example, thetablet or pill can comprise an inner composition covered by an outercomponent. Furthermore, the two components can be separated by anenteric layer that serves to resist disintegration and permits the innercomponent to pass intact through the stomach or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, including a number of polymeric acids and mixtures ofpolymeric acids with such materials as shellac, cetyl alcohol andcellulose acetate.

Pharmaceutical formulations include antagonists (e.g., Wnt-bindingagents) of the present invention complexed with liposomes (Epstein etal., 1985, PNAS, 82:3688; Hwang et al.,1980, PNAS, 77:4030; and U.S.Pat. Nos. 4,485,045 and 4,544,545). Liposomes with enhanced circulationtime are disclosed in U.S. Pat. No. 5,013,556. Liposomes can begenerated by the reverse phase evaporation with a lipid compositioncomprising phosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter.

The antagonist (e.g. Wnt-binding agent) can also be entrapped inmicrocapsules. Such microcapsules are prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions asdescribed in Remington: The Science and Practice of Pharmacy, 21^(st)Edition, University of the Sciences, Philadelphia, 2005.

In addition, sustained-release preparations can be prepared. Suitableexamples of sustained-release preparations include semipermeablematrices of solid hydrophobic polymers containing the agent, whichmatrices are in the form of shaped articles (e.g., films ormicrocapsules). Examples of sustained-release matrices includepolyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and 7 ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), sucrose acetateisobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

V. Methods of Use

The Wnt-binding agents (including soluble receptors) of the inventionare useful in a variety of applications including, but not limited to,therapeutic treatment methods, such as the treatment of cancer. Incertain embodiments, the agents are useful for inhibiting Wnt signaling(e.g., canonical Wnt signaling), inhibiting tumor growth, inducingdifferentiation, reducing tumor volume, reducing cancer stem cellfrequency, and/or reducing the tumorigenicity of a tumor. The methods ofuse may be in vitro, ex vivo, or in vivo methods. In certainembodiments, the Wnt-binding agent or polypeptide is an antagonist ofthe one or more human Wnt proteins to which it binds.

In certain embodiments, the Wnt-binding agents or antagonists are usedin the treatment of a disease associated with Wnt signaling activation.In particular embodiments, the disease is a disease dependent upon Wntsignaling. In particular embodiments, the Wnt signaling is canonical Wntsignaling. In certain embodiments, the Wnt-binding agents or antagonistsare used in the treatment of disorders characterized by increased levelsof stem cells and/or progenitor cells.

In certain embodiments, the disease treated with the Wnt-binding agentor antagonist (e.g., a soluble FZD receptor, SFRP-derived protein, orsoluble Ror receptor) is a cancer. In certain embodiments, the cancer ischaracterized by Wnt-dependent tumors. In certain embodiments, thecancer is characterized by tumors expressing the one or more Wnts towhich the Wnt-binding agent (e.g., soluble receptor) binds.

In certain embodiments, the disease treated with the Wnt-binding agentor antagonist is not a cancer. For example, the disease may be ametabolic disorder such as obesity or diabetes (e.g., type II diabetes)(Jin T., 2008, Diabetologia, 51:1771-80). Alternatively, the disease maybe a bone disorder such as osteoporosis, osteoarthritis, or rheumatoidarthritis (Corr M., 2008, Nat. Clin. Pract. Rheumatol., 4:550-6; Day etal., 2008, Bone Joint Surg. Am., 90 Suppl 1:19-24). The disease may alsobe a kidney disorder, such as a polycystic kidney disease (Harris etal., 2009, Ann. Rev. Med., 60:321-37; Schmidt-Ott et al., 2008, KidneyInt., 74:1004-8; Benzing et al., 2007, J. Am. Soc. Nephroi.,18:1389-98). Alternatively, eye disorders including, but not limited to,macular degeneration and familial exudative vitreoretinopathy may betreated (Lad et al., 2009, Stem Cells Dev., 18:7-16). Cardiovasculardisorders, including myocardial infarction, atherosclerosis, and valvedisorders, may also be treated (Al-Aly Z., 2008, Transl. Res.,151:233-9; Kobayashi et al., 2009, Nat. Cell Biol., 11:46-55; van Gijnet al., 2002, Cardiovasc. Res., 55:16-24; Christman et al., 2008, Am. J.Physiol. Heart Circ. Physiol., 294:H2864-70). In some embodiments, thedisease is a pulmonary disorder such as idiopathic pulmonary arterialhypertension or pulmonary fibrosis (Laumanns et al., 2008, Am. J.Respir. Cell Mol. Biol., 2009, 40:683-91; Königshoff et al., 2008 PLoSONE, 3:e2142). In some embodiments, the disease treated with theWnt-binding agent is a liver disease, such as cirrhosis or liverfibrosis (Cheng et al., 2008, Am. J. Physiol. Gastrointest. LiverPhysiol., 294:G39-49).

The present invention provides methods of treating cancer comprisingadministering a therapeutically effective amount of a Wnt-binding agentto a subject (e.g., a subject in need of treatment). In certainembodiments, the cancer is a cancer selected from the group consistingof colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer,liver cancer, breast cancer, kidney cancer, prostate cancer,gastrointestinal cancer, melanoma, cervical cancer, bladder cancer,glioblastoma, and head and neck cancer. In certain embodiments, thecancer is pancreatic cancer. In certain embodiments, the cancer iscolorectal cancer. In certain embodiments, the cancer is breast cancer.In certain embodiments, the subject is a human.

The present invention further provides methods for inhibiting tumorgrowth using the Wnt-binding agents described herein. In certainembodiments, the method of inhibiting tumor growth comprises contactingthe tumor or tumor cell with a Wnt-binding agent in vitro. For example,an immortalized cell line or a cancer cell line that expresses thetargeted Wnt(s) is cultured in medium to which is added the Wnt-bindingagent to inhibit tumor cell growth. In some embodiments, tumor cells areisolated from a patient sample such as, for example, a tissue biopsy,pleural effusion, or blood sample and cultured in medium to which isadded a Wnt-binding agent to inhibit tumor cell growth.

In some embodiments, the method of inhibiting tumor growth comprisescontacting the tumor or tumor cells with the Wnt-binding agent (e.g., aFZD soluble receptor) in vivo. In certain embodiments, contacting atumor or tumor cell with a Wnt-binding agent is undertaken in an animalmodel. For example, Wnt-binding agents may be administered to xenograftsexpressing one or more Wnts that have been grown in immunocompromisedmice (e.g. NOD/SCID mice) to inhibit tumor growth. In certainembodiments, cancer stem cells are isolated from a patient sample suchas, for example, a tissue biopsy, pleural effusion, or blood sample andinjected into immunocompromised mice that are then administered aWnt-binding agent to inhibit tumor cell growth. In some embodiments, theWnt-binding agent is administered at the same time or shortly afterintroduction of tumorigenic cells into the animal to prevent tumorgrowth. In some embodiments, the Wnt-binding agent is administered as atherapeutic after the tumorigenic cells have grown to a tumor of aspecified size.

In certain embodiments, the method of inhibiting the growth of a tumorcomprises administering to a subject a therapeutically effective amountof a Wnt-binding agent. In certain embodiments, the subject is a human.In certain embodiments, the subject has a tumor or has had a tumorremoved.

The invention also provides methods of reducing cancer stem cellfrequency in a tumor comprising cancer stem cells, the method comprisingadministering a therapeutically effective amount of a Wnt-binding agentto a subject. In addition are provided methods of inducingdifferentiation of tumor cells in a subject, wherein the methodcomprises administering a therapeutically effective amount of aWnt-binding agent to the subject. In some embodiments, methods forinducing expression of differentiation markers in a tumor compriseadministering a therapeutically effective amount of a Wnt-binding agentto a subject. In certain embodiments, the subject is a human.

In certain embodiments, the tumor is a tumor in which Wnt signaling isactive. In certain embodiments, the Wnt signaling that is active iscanonical Wnt signaling. In certain embodiments, the tumor is aWnt-dependent tumor. For example, in some embodiments, the tumor issensitive to axin over-expression. In certain embodiments, the tumordoes not comprise an inactivating mutation (e.g., a truncating mutation)in the adenomatous polyposis coli (APC) tumor suppressor gene or anactivating mutation in the beta-catenin gene. In certain embodiments,the tumor expresses one or more genes in a Wnt gene signature. Incertain embodiments, the cancer for which a subject is being treatedinvolves such a tumor.

In certain embodiments, the tumor expresses the one or more human Wntproteins to which the Wnt-binding agent binds. In certain embodiments,the tumor over-expresses the human Wnt(s).

In certain embodiments, the tumor is a tumor selected from the groupconsisting of colorectal tumor, pancreatic tumor, lung tumor, ovariantumor, liver tumor, breast tumor, kidney tumor, prostate tumor,gastrointestinal tumor, melanoma, cervical tumor, bladder tumor,glioblastoma, and head and neck tumor. In certain embodiments, the tumoris a colorectal tumor. In certain embodiments, the tumor is a pancreatictumor. In certain embodiments, the tumor is a breast tumor.

The invention also provides a method of inhibiting Wnt signaling in acell comprising contacting the cell with an effective amount of aWnt-binding agent. In certain embodiments, the cell is a tumor cell. Incertain embodiments, the method is an in vivo method wherein the step ofcontacting the cell with the agent comprises administering atherapeutically effective amount of the agent to the subject. In somealternative embodiments, the method is an in vitro or ex vivo method. Incertain embodiments, the Wnt signaling that is inhibited is canonicalWnt signaling. In certain embodiments, the Wnt signaling is signaling byWnt1, Wnt2, Wnt3, Wnt3a, Wnt7a, Wnt7b, and/or Wnt10b. In certainembodiments, the Wnt signaling is signaling by Wnt1, Wnt3a, Wnt7b,and/or Wnt10b.

In addition, the invention provides a method of reducing thetumorigenicity of a tumor in a subject, comprising administering atherapeutically effective amount of a Wnt-binding agent to the subject.In certain embodiments, the tumor comprises cancer stem cells. In someembodiments, the tumorigenicity of a tumor is reduced by reducing thefrequency of cancer stem cells in the tumor. In certain embodiments, thefrequency of cancer stem cells in the tumor is reduced by administrationof the Wnt-binding agent. In certain embodiments, the agent or antibodyis capable of reducing the tumorigenicity of a tumor comprising cancerstem cells in an animal model, such as a mouse xenograft model. Incertain embodiments, the number or frequency of cancer stem cells in atumor is reduced by at least about two-fold, about three-fold, aboutfive-fold, about ten-fold, about 50-fold, about 100-fold, or about1000-fold. In certain embodiments, the reduction in the number orfrequency of cancer stem cells is determined by limiting dilution assayusing an animal model. Additional examples and guidance regarding theuse of limiting dilution assays to determine a reduction in the numberor frequency of cancer stem cells in a tumor can be found, e.g., inInternational Publication Number WO 2008/042236, U.S. Patent ApplicationPublication No. 2008/0064049, and U.S. Patent Application PublicationNo. 2008/0178305, each of which is incorporated by reference herein inits entirety.

Thus, the invention also provides a method of reducing the frequency ofcancer stem cells in a tumor comprising cancer stem cells, the methodcomprising contacting the tumor with an effective amount of aWnt-binding agent (e.g., a soluble FZD receptor, a soluble Ror receptoror a SFRP-Fc fusion).

The invention further provides methods of differentiating tumorigeniccells into non-tumorigenic cells comprising contacting the tumorigeniccells with a Wnt-binding agent (for example, by administering theWnt-binding agent to a subject that has a tumor comprising thetumorigenic cells or that has had such a tumor removed). In certainembodiments, the tumorigenic cells are pancreatic tumor cells. Incertain alternative embodiments, the tumorigenic cells are colon tumorcells.

The use of the Wnt-binding agents described herein to induce thedifferentiation of cells, including, but not limited to tumor cells, isalso provided. For example, methods of inducing cells to differentiatecomprising contacting the cells with an effective amount of aWnt-binding agent (e.g., a soluble FZD receptor, a soluble Ror receptor,or a SFRP-Fc fusion) described herein are envisioned. Methods ofinducing cells in a tumor in a subject to differentiate comprisingadministering a therapeutically effective amount of a Wnt-binding agentto the subject are also provided. In certain embodiments, the tumor is apancreatic tumor. In certain other embodiments, the tumor is a colontumor.

Methods of treating a disease or disorder in a subject, wherein thedisease or disorder is associated with Wnt signaling activation and/oris characterized by an increased level of stem cells and/or progenitorcells are further provided. In some embodiments, the treatment methodscomprise administering a therapeutically effective amount of theWnt-binding agent to the subject. In certain embodiments, the Wntsignaling is canonical Wnt signaling.

The Wnt-binding agents or antagonists are administered as an appropriatepharmaceutical composition to a human patient according to knownmethods. Suitable methods of administration include, but are not limitedto, intravenous (administration as a bolus or by continuous infusionover a period of time), intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

In certain embodiments, in addition to administering the Wnt-bindingagent, the method or treatment further comprises administering a secondtherapeutic agent (e.g. an anti-cancer agent) prior to, concurrentlywith, and/or subsequently to administration of the Wnt-binding agent.Pharmaceutical compositions comprising the Wnt-binding agent and thesecond therapeutic agent are also provided.

It will be appreciated that the combination of a Wnt-binding agent and asecond therapeutic agent may be administered in any order orconcurrently. In selected embodiments, the Wnt-binding agents will beadministered to patients that have previously undergone treatment withthe second therapeutic agent. In certain other embodiments, theWnt-binding agent and the second therapeutic agent will be administeredsubstantially simultaneously or concurrently. For example, a subject maybe given the Wnt-binding agent while undergoing a course of treatmentwith the second therapeutic agent (e.g., chemotherapy). In certainembodiments, the Wnt-binding agent will be administered within 1 year ofthe treatment with the second therapeutic agent. In certain alternativeembodiments, the Wnt-binding agent will be administered within 10, 8, 6,4, or 2 months of any treatment with the second therapeutic agent. Incertain other embodiments, the Wnt-binding agent will be administeredwithin 4, 3, 2, or 1 week of any treatment with the second therapeuticagent. In some embodiments, the Wnt -binding agent will be administeredwithin 5, 4, 3, 2, or 1 days of any treatment with the secondtherapeutic agent. It will further be appreciated that the two agents ortreatment may be administered to the subject within a matter of hours orminutes (i.e., substantially simultaneously).

Combination therapy with at least two therapeutic agents often usesagents that work by different mechanisms of action, although this is notrequired. Combination therapy using agents with different mechanisms ofaction may result in additive or synergetic effects. Combination therapymay allow for a lower dose of each agent than is used in monotherapy,thereby reducing toxic side effects. Combination therapy may decreasethe likelihood that resistant cancer cells will develop. In someembodiments, combination therapy comprises a therapeutic agent thataffects (e.g., inhibits or kills) non-tumorigenic cells and atherapeutic agent that affects (e.g., inhibits or kills) tumorigenicCSCs.

Useful classes of therapeutic (e.g., anti-cancer) agents include, forexample, antitubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosureas,platinols, performing compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like. In certain embodiments, the secondtherapeutic agent is an antimetabolite, an antimitotic, a topoisomeraseinhibitor, or an angiogenesis inhibitor.

Therapeutic agents that may be administered in combination with theWnt-binding agents include chemotherapeutic agents. Thus, in someembodiments, the method or treatment involves the combinedadministration of a Wnt-binding agent of the present invention and achemotherapeutic agent or cocktail of multiple differentchemotherapeutic agents. Treatment with a Wnt-binding agent can occurprior to, concurrently with, or subsequent to administration ofchemotherapies. Chemotherapies contemplated by the invention includechemical substances or drugs which are known in the art and arecommercially available, such as gemcitabine, irinotecan, doxorubicin,5-fluorouracil, cytosine arabinoside (“Ara-C”), cyclophosphamide,thiotepa, busulfan, cytoxin, paclitaxel, methotrexate, cisplatin,melphalan, vinblastine, and carboplatin. Combined administration caninclude co-administration, either in a single pharmaceutical formulationor using separate formulations, or consecutive administration in eitherorder but generally within a time period such that all active agents canexert their biological activities simultaneously. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for such chemotherapy arealso described in Chemotherapy Service Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md. (1992).

Chemotherapeutic agents useful in the instant invention also include,but are not limited to, alkylating agents such as thiotepa andcyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan, andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamime; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.paclitaxel (TAXOL) and docetaxel (TAXOTERE), chlorambucil; gemcitabine;6-thioguanine; mercaptopurine; methotrexate; platinum analogs such ascisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine;navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda;ibandronate; CPT11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above. Chemotherapeutic agents also includeanti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens including for example tamoxifen,raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston);and antiandrogens such as flutamide, nilutamide, bicalutamide,leuprolide, and goserelin; and pharmaceutically acceptable salts, acidsor derivatives of any of the above.

In certain embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerase inhibitors are chemotherapy agents thatinterfere with the action of a topoisomerase enzyme (e.g., topoisomeraseI or II). Topoisomerase inhibitors include, but are not limited to,doxorubicin HCl, daunorubicin citrate, mitoxantrone HC1, actinomycin D,etoposide, topotecan HCl, teniposide (VM-26), and irinotecan. In certainembodiments, the second therapeutic agent is irinotecan. In certainembodiments, the tumor to be treated is a colorectal tumor and thesecond therapeutic agent is a topoisomerase inhibitor, such asirinotecan. In some embodiments, the Wnt-binding agent comprises SEQ IDNO:53 and the second therapeutic agent is irinotecan. In someembodiments, the Wnt-binding agent comprises SEQ ID NO:75 and the secondtherapeutic agent is irinotecan.

In certain embodiments, the chemotherapeutic agent is ananti-metabolite. An anti-metabolite is a chemical with a structure thatis similar to a metabolite required for normal biochemical reactions,yet different enough to interfere with one or more normal functions ofcells, such as cell division. Anti-metabolites include, but are notlimited to, gemcitabine, fluorouracil, capecitabine, methotrexatesodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside,thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine,6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, aswell as pharmaceutically acceptable salts, acids, or derivatives of anyof these. In certain embodiments, the second therapeutic agent isgemcitabine. In certain embodiments, the tumor to be treated is apancreatic tumor and the second therapeutic agent is an anti-metabolite(e.g., gemcitabine). In some embodiments, the Wnt-binding agentcomprises SEQ ID NO:53 and the second therapeutic agent is gemcitabine.In some embodiments, the Wnt-binding agent comprises SEQ ID NO:75 andthe second therapeutic agent is gemcitabine.

In certain embodiments, the chemotherapeutic agent is an antimitoticagent, including, but not limited to, agents that bind tubulin. By wayof non-limiting example, the agent comprises a taxane. In certainembodiments, the agent comprises paclitaxel or docetaxel, or apharmaceutically acceptable salt, acid, or derivative of paclitaxel ordocetaxel. In certain embodiments, the agent is paclitaxel (TAXOL),docetaxel (TAXOTERE), albumin-bound paclitaxel (ABRAXANE),DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments,the antimitotic agent comprises a vinka alkaloid, such as vincristine,binblastine, vinorelbine, or vindesine, or pharmaceutically acceptablesalts, acids, or derivatives thereof. In some embodiments, theantimitotic agent is an inhibitor of Eg5 kinesin or an inhibitor of amitotic kinase such as Aurora A or Plk1. In certain embodiments wherethe chemotherapeutic agent administered in combination with theWnt-binding agent or polypeptide comprises an antimitotic agent, thecancer or tumor being treated is breast cancer or a breast tumor. Incertain embodiments, the tumor to be treated is a breast tumor and thesecond therapeutic agent is paclitaxel. In some embodiments, theWnt-binding agent comprises SEQ ID NO:53 and the second therapeuticagent is paclitaxel. In some embodiments, the Wnt-binding agentcomprises SEQ ID NO:75 and the second therapeutic agent is paclitaxel.

In certain embodiments, the treatment involves the combinedadministration of a Wnt-binding agent of the present invention andradiation therapy. Treatment with the Wnt-binding agent can occur priorto, concurrently with, or subsequent to administration of radiationtherapy. Any dosing schedules for such radiation therapy can be used asdetermined by the skilled practitioner.

In some embodiments, the second therapeutic agent comprises an antibody.Thus, treatment can involve the combined administration of Wnt-bindingagents of the present invention with antibodies against tumor-associatedantigens including, but not limited to, antibodies that bind to EGFR,ErbB2, HER2, DLL4, Notch, and/or VEGF. Exemplary, anti-DLL4 antibodiesare described, for example, in U.S. Pat. No. 7,750,124, incorporated byreference herein in its entirety. Additional anti-DLL4 antibodies aredescribed in, e.g., International Patent Publication Nos. WO 2008/091222and WO 2008/0793326, and U.S. Patent Application Publication Nos. US2008/0014196, US 2008/0175847, US 2008/0181899, and US 2008/0107648,each of which is incorporated by reference herein in its entirety. Incertain embodiments, the second therapeutic agent is an antibody that isan angiogenesis inhibitor (e.g., an anti-VEGF antibody). In someembodiments, the Wnt-binding agent comprises SEQ ID NO:53 and the secondtherapeutic agent is an anti-VEGF antibody. In some embodiments, theWnt-binding agent comprises SEQ ID NO:75 and the second therapeuticagent is an anti-VEGF antibody. In certain embodiments, the secondtherapeutic agent is an inhibitor of Notch signaling. In someembodiments, the second therapeutic agent is an anti-Notch antibody.Exemplary anti-Notch antibodies are described, for example, in U.S.Patent Application Publication No. US 2008/0131434, incorporated byreference herein in its entirety. In certain embodiments, the secondtherapeutic agent is bevacizumab (AVASTIN), trastuzumab (HERCEPTIN),panitumumab (VECTIBIX), or cetuximab (ERBITUX). Combined administrationcan include co-administration, either in a single pharmaceuticalformulation or using separate formulations, or consecutiveadministration in either order but generally within a time period suchthat all active agents can exert their biological activitiessimultaneously.

Furthermore, treatment can include administration of one or morecytokines (e.g., lymphokines, interleukins, tumor necrosis factors,and/or growth factors) or can be accompanied by surgical removal oftumor or cancer cells or any other therapy deemed necessary by atreating physician.

For the treatment of the disease, the appropriate dosage of an agent ofthe present invention depends on the type of disease to be treated, theseverity and course of the disease, the responsiveness of the disease,whether the agent is administered for therapeutic or preventativepurposes, previous therapy, patient's clinical history, and so on all atthe discretion of the treating physician. The agent can be administeredone time or over a series of treatments lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved (e.g. reduction in tumor size). Optimal dosingschedules can be calculated from measurements of drug accumulation inthe body of the patient and will vary depending on the relative potencyof an individual agent. The administering physician can easily determineoptimum dosages, dosing methodologies and repetition rates. In certainembodiments, dosage is from 0.01 μg to 100 mg per kg of body weight, andcan be given once or more daily, weekly, monthly or yearly. In certainembodiments, the dosage of the soluble receptor or other Wnt-bindingagent is from about 0.1 mg to about 20 mg per kg of body weight. Incertain embodiments, the Wnt-binding agent is given once every week. Incertain embodiments, the Wnt-binding agent is given once every two weeksor once every three weeks. The treating physician can estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues.

The present invention further provides methods of screening agents forefficacy in inhibiting Wnt signaling, for anti-tumor activity, and/oractivity against cancer stem cells. In certain embodiments, the methodcomprises comparing the level of one or more differentiation markersand/or one or more sternness markers in a first solid tumor (e.g., asolid tumor comprising cancer stem cells) that has been exposed to theagent to the level of the one or more differentiation markers in asecond solid tumor that has not been exposed to the agent. In someembodiments, the method comprises: (a) exposing a first solid tumor, butnot a second solid tumor, to the agent; (b) assessing the level of oneor more differentiation markers and/or one or more sternness markers inthe first and second solid tumors; and (c) comparing the level of theone or more differentiation markers in the first tumor and the level ofthe one or more differentiation markers in the second solid tumor. Incertain embodiments, the (a) increased levels of the one or moredifferentiation markers in the first solid tumor relative to the levelsof the one or more differentiation markers in the second solid tumorindicates anti-tumor (or anti-cancer stem cell) activity; and (b)decreased levels of the one or more sternness markers indicateanti-tumor (or anti-cancer stem cell) activity. In certain embodiments,the agent binds one or more Wnt proteins. In certain embodiments, theagent is a FZD soluble receptor. In certain methods, the agent is anantibody, such as an anti-Wnt antibody.

Additional methods for screening agents include, but are not limited to,methods comprising comparing the levels of one or more differentiationmarkers in a first solid tumor that has been exposed to an agent to thelevels of the one or more differentiation markers in a second solidtumor that has not been exposed to the agent. In certain embodiments,the methods include comprising (a) exposing a first solid tumor, but nota second solid tumor, to the agent; (b) assessing the levels of one ormore differentiation markers in the first and second solid tumors; and(c) comparing the levels of the one or more differentiation markers inthe first tumor to the levels of the one or more differentiation markersin the second solid tumor. In certain embodiments, the agent is aWnt-binding agent. In certain embodiments, the agent is an inhibitor ofthe canonical Wnt signaling pathway. In certain embodiments, the agentinhibits binding of one or more human Wnt proteins to one or more humanFZD receptors. In certain embodiments, increased levels of one or moredifferentiation markers in the first solid tumor relative to levels ofone or more differentiation markers in the second solid tumor indicatesefficacy against solid tumor stem cells (CSCs). In certain alternativeembodiments, decreased levels of one or more differentiation markers(i.e., negative markers for differentiation) in the first solid tumorrelative to the levels of one or more differentiation markers in thesecond solid tumor indicates efficacy against solid tumor stem cells.

In certain embodiments, the solid tumor in the screening method is apancreatic tumor. In certain embodiments, the solid tumor is apancreatic tumor and the one or more differentiation markers maycomprise one or more mucins (e.g., Muc16), one or more cytokeratins(e.g., CK20) and/or chromogranin A (CHGA).

In certain alternative embodiments, the solid tumor in the screeningmethod is a colon tumor. In some embodiments, the solid tumor is a colontumor and the one or more differentiation markers may comprise one ormore cytokeratins (e.g., cytokeratin 7 or CK20).

In certain embodiments, the one or more stemness markers used in thescreening methods described herein comprise ALDH1A1, APC, AXIN2, BMI1,CD44, FGF1, GJB1, GJB2, HES1, JAG1, LGR5, LHX8, MYC, NANOG, NEUROD1,NEUROG2, NOTCH1, NOTCH2, NOTCH3, NOTCH4, PROCR, RARRES1, RARRES3, RBP2,SOX1, SOX2, ASCL2,TDGF1, OLFM4, MSI1, DASH1, EPHB3 and/or EPHB4. Incertain embodiments, two or more sternness markers, three or moresternness markers, four or more stemness markers, five or more sternnessmarkers, six or more, or ten or more stemness markers are selected fromthe group consisting of ALDH1A1, APC, AXIN2, BMI1, CD44, FGF1, GJB1,GJB2, HES1, JAG1, LGR5, LHX8, MYC, NANOG, NEUROD1, NEUROG2, NOTCH1,NOTCH2, NOTCH3, NOTCH4, PROCR, RARRES1, RARRES3, RBP2, SOX1, SOX2,ASCL2,TDGF1, OLFM4, MSI1, DASH1, EPHB3 and EPHB4.

In certain embodiments, the one or more differentiation markers used inthe screening methods comprise ALDOB, BMP2, BMP7, BMPR1B, CEACAM5,CEACAM6, CDX1, CDX2, CLCA2, COL1A2, COL6A1, CHGA, CSTA, CST4, CK20,DAB2, FABP4, GST1, KRT4, KRT7, KRT15, KRT17, KRT20, LAMA1, MUC3A, MUC4,MUC5AC, MUC5B, MUC13, MUC15, MUC16, MUC17, NDRG2, PIP, PLUNC, SPRR1A,REG4, VSIG1, and/or XAF1. In certain embodiments two or more, three ormore, four or more, five or more, six or more, or ten or moredifferentiation markers used in the screening methods are selected fromthe group consisting of ALDOB, BMP2, BMP7, BMPR1B, CEACAM5, CEACAM6,CDX1, CDX2, CLCA2, COL1A2, COL6A1, CHGA, CSTA, CST4, CK20, DAB2, FABP4,GST1, KRT4, KRT7, KRT15, KRT17, KRT20, LAMA1, MUC3A, MUC4, MUC5AC,MUC5B, MUC13, MUC15, MUC16, MUC17, NDRG2, PIP, PLUNC, SPRR1A, REG4,VSIG1, and XAF 1.

Other potential differentiation markers for pancreas and colon as wellas other tumor types are known to those skilled in the art. In addition,the usefulness of potential differentiation markers in a screeningmethod can be readily assessed by one skilled in the art by treating thedesired tumor type with one or more of the soluble FZD receptorsdescribe herein such as FZD8-Fc and then assessing for changes inexpression of the marker by the treated tumor relative to control.Non-limiting examples of such methods, can for instance, be found in thespecific Examples below.

The present invention further provides methods for producing solubleWnt-binding agents. In certain embodiments, the method comprisesproducing a soluble Wnt-binding agent which comprises a Fri domain ofhuman FZD8 in a cell, wherein at least 80% of the Wnt-binding agent hasan N-terminal sequence of ASA, the method comprising using a signalsequence selected from the group consisting of: SEQ ID NO:71, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74 for production ofthe Wnt-binding agent. In some embodiments of the method, the signalsequence is SEQ ID NO:71. In some embodiments, at least about 90%, atleast about 95%, or at least about 98% of the Wnt-binding agent has anN-terminal sequence of ASA. In some embodiments, the cell is a mammaliancell. In some embodiments, the Wnt-binding agent comprises a human Fcregion. In some embodiments, the Wnt-binding agent comprises an aminoacid sequence selected from the group consisting of: SEQ ID NO:53, SEQID NO:50, SEQ ID NO:46, SEQ ID NO:48, and SEQ ID NO:1. In someembodiments, the Wnt-binding agent comprises SEQ ID NO:53. In someembodiments, the Wnt-binding agent comprises SEQ ID NO:50. In someembodiments, the cell comprises a polynucleotide comprising apolynucleotide that encodes a polypeptide having the sequence of SEQ IDNO:75.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application.

Example 1 Production of FZD8-Fc

cDNA encoding FZD8-Fc (54F03) was subcloned into a pEE14.4 expressionvector (Lonza) digested with HindIII and EcoRI. After cloning, thepEE14.4-FZD8-Fc DNA was linearized by digestion with PvuI andsubsequently introduced into GS-CHOK1 cells by electroporation usingstandard procedures. Stable clones expressing FZD8-Fc were obtained, andexpanded in serum free medium. FZD8-Fc was purified by affinity captureusing a protein A-conjugated resin. SDS-PAGE analysis revealed greaterthan 98% purity and endotoxin level was lower than 1 EU/mg protein.

The amino acid sequence of FZD8-Fc is SEQ ID NO:1 and the polynucleotidesequence encoding FZD8-Fc is SEQ ID NO:2.

Example 2 Pharmacokinetics of FZD8-Fc in Rat

The pharmacokinetics of FZD8-Fc (54F03) were assessed in rats in a twoweek pharmacokinetics (PK) study using doses of 2 mg/kg and 10 mg/kg.Sprague Dawley rats, five males in each group, were dosed with FZD8-Fcvia the tail vein at 2 mg/kg or 10 mg/kg and followed for two weeks withsamples collected at the time points 1, 24, 48, 72, 96, 168, 240, and336 hours. At each time point, lml of blood was collected intopotassium-EDTA tubes and centrifuged. The plasma supernatants werecollected and frozen until the samples were analyzed.

The level of FZD8-Fc fusion protein present in the plasma at each timepoint was quantified and the half-life of FZD8-Fc was calculated for thetwo doses. As shown in FIG. 1, the half-life of FZD8-Fc was estimated tobe 163 hours at 2 mg/kg and to be 157 hours at 10 mg/kg.

Example 3 Anti-Tumor Activity of FZD8-Fc in Pancreatic Tumor Model

Inhibition of tumor growth by FZD8-Fc in pancreatic tumor model. Theanti-tumor activity of FZD8-Fc (54F03) was evaluated in the PN4 pancreastumor xenograft model. Dissociated OMP-PN4 cells (50,000 per animal)were injected subcutaneously into 6-8 week old male NOD/SCID mice. Tumorgrowth was monitored weekly and tumor measurements were initiated oncetumors were palpable. On day 50, mice with average tumor volumes of 137mm³ were randomized into 4 groups of 10 animals each. Animals wereinjected with either control antibody, FZD8-Fc (15 mg/kg), gemcitabine(2 mg/kg) or a combination of FZD8-Fc and gemcitabine. Administration ofthe FZD8-Fc and gemcitabine was performed via injection into theintra-peritoneal cavity, once weekly (gemcitabine) or twice weekly(FZD8-Fc). Tumors were measured twice a week and tumor volume wasdetermined using the formula ½(a×b²); where a=length, and b=breadth.Data are expressed as mean and mean±S.E.M. Group means were comparedusing Student's two-tailed, unpaired t test. Probability (p) values of<0.05 were interpreted as significantly different.

Treatment with FZD8-Fc resulted in a 66% reduction in tumor growth, asshown in FIG. 2 (p<0.001). Furthermore, treatment with FZD8-Fc andgemcitabine resulted in a 29% reduction of tumor growth relative totreatment with FZD8-Fc alone (p=0.04 vs. FZD8-Fc alone) (FIG. 2). Thus,FZD8-Fc demonstrated anti-tumor growth activity in the PN4 pancreastumor model as a single agent as well as in combination withgemcitabine.

Reduction of CD44hi population in PN4 tumors treated with FZD8-Fc.Control and treated tumors from the OMP-PN4 xenograft study describedabove were harvested at the end of the study (day 85). The tumors wereprocessed and dissociated into single cells. Single cell suspensionsderived from 5 tumors of each treatment group were pooled, and thepooled samples were then incubated on ice for 30 min with antibodiesthat bind mouse cells selectively (a-mouse CD45-biotin 1:200 dilutionand rat α-mouse H2Kd-biotin 1:100 dilution, BioLegend, San Diego,Calif.), followed by addition of streptavidin-labeled magnetic beads(Invitrogen, Carlsbad, Calif.). Mouse cells were removed with the aid ofa magnet. For analysis of human cell surface markers, the single tumorcell suspension was stained with anti-ESA (Biomeda, Hayward, Calif.) andanti-CD44 (BD Biosciences, San Jose, Calif.) antibodies which weredirectly conjugated to fluorochromes. Dead cells were excluded by usingthe viability dye DAPI. Flow cytometry was performed using a FACS Ariainstrument (Becton Dickinson, Franklin Lakes, N.J.). Side scatter andforward scatter profiles were used to eliminate cell clumps.

Analysis of the tumors treated with control antibody revealed that 12.7%of the bulk tumor population expressed both ESA and CD44 at high levels.The double positive population was not significantly affected bytreatment with gemcitabine alone (13.9%) as shown in FIG. 3, buttreatment with either FZD8-Fc or the combination of FZD8-Fc withgemcitabine reduced the double positive population (1.9% and 1.7%respectively).

Analysis of FZD8-Fc-treated PN4 tumors by limiting dilution assay.Limiting dilution assays (LDA) can be used to assess the effect oftherapeutic agents on solid tumor cancer stem cells and on thetumorigenicity of a tumor comprising the cancer stem cells. Such assayscan be used to determine the frequency of cancer stem cells in tumorsfrom animals treated with the FZD8-Fc fusion protein or other agent andto compare that frequency to the frequency of cancer stem cells intumors from control animals.

Control and treated tumors from the PN4 xenograft study described abovewere harvested at the end of the study (day 85). The tumors wereprocessed and dissociated into single cells. Single cell suspensionsderived from 5 tumors of each treatment group were pooled, and thepooled samples were then incubated on ice for 30 min with antibodiesthat bind mouse cells selectively (α-mouse CD45-biotin 1:200 dilutionand rat a-mouse H2Kd-biotin 1:100 dilution, BioLegend, San Diego,Calif.), followed by addition of streptavidin-labeled magnetic beads(Invitrogen, Carlsbad, Calif.). The mouse cells were removed with theaid of a magnet. The human cells in the suspension were harvested,counted, and stained for cell surface markers and appropriate cell doses(30, 90, and 270 cells) in FACS buffer were mixed in a 1:1 mixture withMatrigel and injected subcutaneously in NOD/SCID mice (10 mice per celldose per treatment group). Tumors are allowed to grow for up to 4months.

At the desired time point, the percentage of mice with detectable tumorswas determined in all groups injected with FZD8-Fc-treated tumor cellsand compared to the percentage of mice with detectable tumors in thecontrols. For example, the number of mice injected with 125 controlantibody-treated tumor cells that have detectable tumors is determinedand compared to the number of mice injected with 125 FZD8-Fc treatedtumor cells that have detectable tumors.

On day 75 after injection of the cells, tumor take rates in the variousgroups were as follows: control—7 mice out of 30 mice; FZD8-Fc—3 miceout of 30 mice; gemcitabine—7 mice out of 30 mice; FZD8-Fc andgemcitabine—0 mice out of 30 mice (FIG. 4). The reduced tumor take ratein the FZD8-Fc and in the combination treated groups indicated that thecancer stem cell frequency was reduced in PN4 pancreatic tumors byFZD8-Fc. The evidence from the assessment of both, CD44 expression andlimiting dose dilution analysis revealed that FZD8-Fc treatment reducescancer stem cell frequency in PN4 pancreatic tumors.

The cancer stem cell (CSC) frequency can be calculated using L-Calc™software (StemCell Technologies Inc.; www.stemcell.com). Briefly, basedon Poisson statistics, exactly one cancer stem cell exists among theknown number of injected cells if 37% of the animals fail to developtumors. The CSC frequency for the control antibody treated group was1:280, the CSC frequency for the gemcitabine treated group was 1:476,the CSC frequency for the FZD8-Fc treated group was 1:881, and the CSCfrequency for the group treated with a combination of FZD8-Fc andgemcitabine was calculated to be lower than 1:3763. This number couldnot be accurately determined because the tumor take rate in this groupwas zero, even at the highest cell dose.

Example 4 Increased Cell Differentiation of Pancreatic Tumors by FZD8-Fc

Increased cell differentiation of PN4 and PN8 tumors with FZD8-Fctreatment. Control and treated tumors from the OMP-PN4 xenograft studydescribed above (Example 3) were harvested at the end of the study (day85). Tumors were fixed in formalin, embedded in paraffin, and tumorsections of 4 μm thickness were cut. After deparaffinization andhydration, the sections were treated with aqueous acetic acid for 5minutes at room temperature. The sections were then treated with 1%alcian blue in 3% aqueous acetic acid for 30 minutes and washed withwater. Sections were counter-stained in neutral fast red, dehydrated andmounted. Using this method, sialomucins in the tissue samples stain blueand the background appears as pink or red.

The treatment of PN4 tumors with FZD8-Fc caused an increase in cellsexpressing sialomucins as compared to tumors treated with controlantibody or gemcitabine (FIG. 5, where the sialomucins appear as a darkgray). The combination treatment of FZD8-Fc and gemcitabine alsoincreased the expression of sialomucins in PN4 pancreatic tumors.Therefore, FZD8-Fc treatment of PN4 tumors increased the frequency ofmucin-expressing differentiated cells. Similar results were seen in thePN8 pancreatic tumor xenograft model (FIG. 6).

Increased cell differentiation of PN13 tumors with FZD8-Fc treatment.The cell differentiation capability of FZD8-Fc was also evaluated in theOMP-PN13 pancreas tumor xenograft model. Dissociated OMP-PN13 cells(50,000 per animal) were injected subcutaneously into 6-8 week old maleNOD/SCID mice. Tumor growth was monitored weekly and tumor measurementswere initiated once tumors were palpable. On day 40 mice with averagetumor volume of 114 mm³ were randomized into 4 groups of 10 animalseach. Animals were injected with either control antibody or FZD8-Fc (15mg/kg). Administration of the FZD8-Fc and control antibody was performedvia injection into the intraperitoneal cavity, twice weekly. After 19days of treatment, the tumors were excised and immunohistochemistryanalysis was performed using standard techniques. Briefly, tumors werefixed in formalin, embedded in paraffin, and tumor sections of 4 μmthickness were cut. After deparaffinization and hydration, the tumorsections were subjected to an antigen retrieval process in Tris buffer(pH 9.5). Sections were incubated with hydrogen peroxide (Sigma-Aldrich,St Louis, Mo.) for 10 minutes to block endogenous peroxidases. Anti-Ki67antibody (Dako, clone MIB-1) at 1:200 dilution in blocking buffer (3%NHS, 1% BSA, 0.1% Tween-20, in PBS) was added to each section andincubated for 1 hour. Slides were rinsed 3 times in PBST for 5 minuteseach. Anti-mouse secondary antibody conjugated with HRP (ImmPRESS™anti-mouse, Vector Laboratories Inc., Burlingame, Calif.) was added tothe slides and incubated for 30 minutes. After multiple washes withPBST, Vector Nova Red substrate (Vector Laboratories Inc., Burlingame,Calif.) was added for localization of Ki67 antigen. The sections weretreated with aqueous acetic acid for 5 minutes at room temperature. Thesections were then treated with 1% alcian blue in 3% aqueous acetic acidfor 30 min and washed with water. Sections were counter-stained inneutral fast red, dehydrated and mounted. Using this method, sialomucinsin the tissue samples stain blue and proliferating cells are marked darkred.

The treatment of PN13 tumors with FZD8-Fc resulted in an increase incells expressing sialomucins as compared to tumors treated with controlantibody. Treatment of PN13 tumors with FZD8-Fc also resulted in adecrease in proliferating cells as denoted by expression of Ki67. FIG. 7shows a clear decrease in the number of proliferating cells (identifiedby black spots) in the tissue from FZD8-Fc treated tumors. Thereforetreatment of PN13 tumors with FZD8-Fc decreased cell proliferation, andincreased frequency of mucin expressing differentiated cells.

Increased Muc16 staining in Pn13 tumors with FZD8-Fc. Tumor sectionsfrom PN13 tumors treated with control antibody or FZD8-Fc were obtainedand treated as described above. In this example, anti-Muc16 (Abcam,Cambridge, Mass.) antibody in blocking buffer at 1:200 dilution (3% NHS,1% BSA, 0.1% Tween-20 in PBS) was added to each section and incubatedfor 1 hour. The bound antibody was detected using theimmunohistochemistry protocol described above.

The treatment of PN13 tumors with FZD8-Fc resulted in an increase incells expressing Muc16 as compared to tumors treated with controlantibody (FIG. 8, dark staining).

Increased CK20 staining in Pn13 tumors with FZD8-Fc. Tumor sections wereobtained and treated as described above. In this example, anti-CK20(clone Ks20.8, Dako, Carpinteria, Calif.) antibody in blocking buffer at1:200 dilution (3% NHS, 1% BSA, 0.1% Tween-20, in PBS) was added to eachsection and incubated for 1 hour. The bound antibody was detected usingthe immunohistochemistry protocol described above.

The treatment of PN13 tumors with FZD8-Fc resulted in an increase incells expressing CK20 as compared to tumors treated with controlantibody (FIG. 9, dark staining).

Example 5 Inhibition of Breast Tumor Growth in vivo by FZD8-Fc

Dissociated PE13 breast tumor cells (50,000 per animal) were injectedsubcutaneously into the mammary fat pads of NOD/SCID mice. Mice weremonitored weekly and tumors were allowed to grow until they wereapproximately 106 mm³. On day 27 post cell injection the mice wererandomized into four treatment groups (n=10 mice/group) and treated withcontrol antibody, FZD8-Fc (54F03), taxol or a combination of FZD8-Fc andtaxol. Taxol was administered intraperitoneally at a dose of 7.5 mg/kgonce a week and FZD8-Fc was administered intraperitoneally at a dose of5 mg/kg twice a week. Tumors were measured on the days indicated in FIG.10.

Treatment with FZD8-Fc was observed to reduce tumor growth by 20%(p=0.002) as a single agent relative to the control antibody group. Inaddition, treatment with the combination of FZD8-Fc and taxol reducedtumor growth by 55% (p=0.003) as compared to taxol treatment alone (FIG.10).

Example 6 Inhibition of Colon Tumor Growth in vivo by FZD8-Fc

The effect of multiple doses and dosing regimen of FZD8-Fc (54F03) onthe growth of C28 colon tumor xenografts was analyzed. Dissociated C28cells (10,000 per animal) were injected subcutaneously into 6-8 week oldmale NOD/SCID mice. On day 2, mice were randomized into 6 groups of 10animals each. Animals were injected with either control antibody orFZD8-Fc at doses of 1.5 mg/kg (twice a week), 5 mg/kg (once and twice aweek) and 15 mg/kg (once and twice a week). Administration of theantibody and FZD8-Fc was performed via injection into theintraperitoneal cavity. Tumor growth was monitored weekly and tumormeasurements were initiated once tumors were palpable. Tumors weremeasured twice a week and tumor volume was determined as describedherein.

Treatment with 15 mg/kg of FZD8-Fc (twice weekly) resulted in 83%reduction in tumor growth over treatment with the control antibody, asshown in FIG. 11A (p<0.001). Furthermore, treatment with FZD8-Fc at thelowest dose evaluated (1.5 mg/kg administered twice a week) alsoresulted in a 52% reduction of growth over control antibody treatmentgroup (FIG. 11A). Thus, FZD8-Fc demonstrated anti-tumor growth activityin the OMP-C28 colon tumor model as a single agent in a dose dependentmanner.

The effect of FZD8-Fc in combination with a chemotherapeutic agent onthe growth of C28 colon tumor xenografts was analyzed. Dissociated C28colon tumor cells (10,000 cells) were injected subcutaneously into 6-8week old male NOD/SCID mice. Tumors were allowed to grow for 21 daysuntil they reached an average volume of 128 mm³. The mice wererandomized (n=10 per group) and treated with FZD8-Fc (54F03) (15 mg/kgonce a week), irinotecan (15 mg/kg once a week), a combination ofFZD8-Fc and irinotecan or a control antibody. Administration of theFZD8-Fc, irinotecan and control antibody was performed via injectioninto the intraperitoneal cavity. Tumor growth was monitored and tumorvolumes were measured with electronic calipers at the indicated timepoints. Data are expressed as mean±S.E.M.

As shown in FIG. 11B, treatment with FZD8-Fc as a single agent (-▴-)resulted in 66% reduction in tumor growth over treatment with thecontrol antibody (-▪-) (p<0.001). Furthermore, treatment with FZD8-Fc incombination with irinotecan (-•-) resulted in a 76% reduction of growthover control antibody treatment group (p<0.001), which was greater thaneither agent alone. Thus, FZD8-Fc demonstrated anti-tumor growthactivity in the OMP-C28 colon tumor model as a single agent, as well asin combination with a chemotherapeutic agent.

Example 7 Increased Cell Differentiation of Colon Tumors by FZD8-Fc

Increased CK20 staining in C28 tumors with FZD8-Fc. Control and treatedtumors from the C28 xenograft study described above (Example 6) wereharvested at the end of the study. The tumors were excised andimmunohistochemistry analysis was performed using standard techniques.Briefly, tumors were fixed in formalin, embedded in paraffin, and tumorsections of 4 um thickness were cut. After deparaffinization andhydration, the tumor sections were subjected to an antigen retrievalprocess in Tris buffer (pH 9.5). Sections were incubated with hydrogenperoxide (Sigma-Aldrich, St Louis, Mo.) for 10 minutes to blockendogenous peroxidases. Anti-CK20 (clone Ks20.8, Dako, Carpinteria,Calif.) antibody in blocking buffer at 1:200 dilution (3% NHS, 1% BSA,0.1% Tween-20, in PBS) was added to each section and incubated for 1hour. Slides were rinsed 3 times in PBST for 5 minutes each. Anti-mousesecondary antibody conjugated with HRP (ImmPRESS™ anti-mouse Ig, VectorLaboratories Inc., Burlingame, Calif.) was added to the slides andincubated for 30 minutes. After multiple washes with PBST, Vector NovaRed substrate (Vector Laboratories Inc., Burlingame, Calif.) was addedfor localization of CK20 antigen.

The treatment of C28 tumors with FZD8-Fc resulted in an increase incells expressing CK20 as compared to tumors treated with controlantibody (FIG. 12, dark staining).

Example 8 Anti-Tumor Activity of FZD8-Fc in Pancreatic Tumor Model

Inhibition of tumor growth by FZD8-Fc in PN21 pancreatic tumor model.The anti-tumor activity of FZD8-Fc (54F03) was evaluated in the PN21pancreas tumor xenograft model. Dissociated OMP-PN21 cells (50,000 peranimal) were injected subcutaneously into 6-8 week old male NOD/SCIDmice. Tumor growth was monitored weekly and tumor measurements wereinitiated once tumors were palpable. On day 36, mice with average tumorvolumes of 144 mm³ were randomized into 4 groups of 9 animals each.Animals were injected with control antibody, FZD8-Fc (15 mg/kg),gemcitabine (2 mg/kg) or a combination of FZD8-Fc and gemcitabine.Administration of the FZD8-Fc and gemcitabine was performed viainjection into the intraperitoneal cavity, once weekly. Tumors weremeasured twice a week and tumor volume was determined as describedherein.

Treatment with FZD8-Fc resulted in a 66% reduction in tumor growth ascompared to control, as shown in FIG. 13A (p<0.001). Furthermore,treatment with a combination of FZD8-Fc and gemcitabine resulted in agreater reduction of tumor growth compared to either agent alone(p=0.001 vs. gemcitabine). Thus, FZD8-Fc demonstrated anti-tumor growthactivity in the PN21 pancreas tumor model as a single agent as well asin combination with gemcitabine.

Analysis of FZD8-Fc-treated PN21 tumors by limiting dilution assay. Asdescribed above in Example 3, a limiting dilution assays was used toassess the effect of treatment with FZD8-Fc alone or in combination withgemcitabine on solid tumor cancer stem cells in the PN21 pancreatictumor model.

Control and treated tumors from the PN21 xenograft study described abovewere harvested at the end of the study. The tumors were processed anddissociated into single cells. Single cell suspensions derived from 5tumors of each treatment group were pooled, and the pooled samples werethen incubated on ice for 30 min with antibodies that bind mouse cellsselectively (α-mouse CD45-biotin 1:200 dilution and rat α-mouseH2Kd-biotin 1:100 dilution, BioLegend, San Diego, Calif.), followed byaddition of streptavidin-labeled magnetic beads (Invitrogen, Carlsbad,Calif.). The mouse cells were removed with the aid of a magnet. Thehuman cells in the suspension were harvested, counted, and stained forcell surface markers and appropriate cell doses (30, 90, and 270 cells)in FACS buffer were mixed in a 1:1 mixture with Matrigel and injectedsubcutaneously in NOD/SCID mice (10 mice per cell dose per treatmentgroup). Tumors are allowed to grow for up to 4 months.

At the desired time point, the percentage of mice with detectable tumorswas determined in all groups injected with treated tumor cells andcompared to the percentage of mice with detectable tumors in controltreated cells. For example, the number of mice injected with 125 controlantibody-treated tumor cells that have detectable tumors is determinedand compared to the number of mice injected with 125 FZD8-Fc treatedtumor cells that have detectable tumors.

On day 72 after injection of the cells, tumor take rates in the variousgroups were determined and the cancer stem cell frequency was calculatedusing L-Calc™ software (StemCell Technologies Inc.; www.stemcell.com).As shown in FIG. 13B, treatment with FZD8-Fc reduced cancer stem cellfrequency to 1:976, approximately a four-fold reduction as compared totreatment with the control antibody. In contrast, treatment withgemcitabine slightly increased cancer stem cell frequency.Significantly, treatment with a combination of FZD8-Fc and gemcitabinereduced the cancer stem cell frequency to 1:5472, almost a 25-foldreduction as compared to treatment with control. Surprisingly, treatmentwith the combination of FZD8-Fc and gemcitabine reduced the cancer stemcell frequency approximately 5.5-fold greater than FZD8-Fc treatmentalone and despite the fact that gemcitabine appeared to actuallyincrease the cancer stem cell frequency.

Example 9

Increased cell differentiation of PN21 tumors with FZD8-Fc. The celldifferentiation capability of FZD8-Fc was also evaluated in the OMP-PN21pancreas tumor xenograft model. PN21 tumors from studies described inExample 8 were harvested and fixed in formalin, embedded in paraffin,and tumor sections of 4 μm thickness were cut. After deparaffinizationand hydration, the tumor sections were subjected to an antigen retrievalprocess in Tris buffer (pH 9.5). Sections were incubated with hydrogenperoxide (Sigma-Aldrich, St Louis, Mo.) for 10 minutes to blockendogenous peroxidases. Anti-Ki67 antibody (clone MIB-1, Dako,Carpinteria, Calif.) in blocking buffer (3% NHS, 1% BSA, 0.1% Tween-20in PBS) at 1:200 dilution was added to each section and incubated for 1hour. Slides were rinsed 3 times in PBST for 5 minutes each. Anti-mousesecondary antibody conjugated with HRP (ImmPRESS™ anti-mouse, VectorLaboratories Inc., Burlingame, Calif.) was added to the slides andincubated for 30 minutes. After multiple washes with PBST, Vector NovaRed substrate (Vector Laboratories Inc., Burlingame, Calif.) was addedfor localization of Ki67 antigen. The sections were treated with aqueousacetic acid for 5 minutes at room temperature. The sections were thentreated with 1% alcian blue in 3% aqueous acetic acid for 30 minutes andwashed with water. Sections were counter-stained in neutral fast red,dehydrated and mounted. Using this method, sialomucins in the tissuesamples stain blue and the background appears as pink or red.

The treatment of PN21 tumors with FZD8-Fc resulted in an increase incells expressing sialomucins as compared to tumors treated with controlantibody (FIG. 14, dark gray staining). The treatment of PN21 tumorswith FZD8-Fc or FZD-Fc in combination with gemcitabine resulted in anincrease in cells expressing sialomucins as compared to tumors treatedwith control antibody or gemcitabine alone (FIG. 15). Treatment of PN21tumors with FZD8-Fc or the combination of FZD8-Fc and gemcitabine alsoresulted in a decrease in proliferating cells denoted by expression ofKi67. Therefore treatment of PN21 tumors with FZD8-Fc, either alone orin combination with gemcitabine, decreased cell proliferation, andincreased frequency of mucin-expressing differentiated cells.

Example 10 Production of FZD8-Fc Variants

Production of FZD8-Fc variants. FZD8-Fc variants were produced at DNA2.0(Menlo Park, Calif.). DNA2.0 synthesized and assembled shortsingle-stranded oligonucleotides to produce the different FZD8-Fcvariant proteins, 54F05, 54F08, 54F09, 54F12, 54F13, 54F14, 54F15,54F16, 54F17, 54F18, 54F19, 54F20, 54F21 and 54F22. The assembledoligonucleotides were subsequently cloned and sequence verified.

Example 11 Inhibition of Wnt Signaling by FZD8-Fc Variants

The ability of the FZD8-Fc variants to block or inhibit activation ofthe Wnt signaling pathway was determined in vitro using a luciferasereporter assay. STF293 cells were cultured in DMEM supplemented withantibiotics and 10% FCS. The STF293 cells are stably transfected with areporter vector containing seven copies of the TCF transcriptionalresponse element linked to a promoter upstream of a firefly luciferasereporter gene. This construct measures the activity of the canonical Wntsignaling pathway. The cells were added to cultures plates at 10,000cells per well. After an overnight incubation the FZD8-Fc variants orcontrol mouse JAG1-Fc were added in combination with Wnt3a-conditionedmedium. The FZD8-Fc variants and JAG1-Fc were used at concentrations of20, 4, 0.8, 0.16, 0.03, 0.006, 0.0012, and 0.0003 ug/ml. The cells wereincubated in 25% Wnt3A-conditioned medium that had been prepared from Lcells that stably express Wnt3a. After overnight incubation(approximately 18 hrs), luciferase levels were measured using aSteady-Glo® luciferase assay kit (Promega, Madison, Wis.).

The blocking activity of the FZD8-Fc variants was determined and ispresented in Table 3 as relative activity as compared to the samereference standard run in each assay which was set at 100%.

TABLE 3 FZD8-Fc Variant % Relative Activity 54F03 107, 143 54F05 16754F08 137 54F09 156, 157 54F12  52 54F13 103 54F14 125 54F15 128 54F16125

Example 12 Pharmacokinetics of FZD8-Fc Variants in Rats

The pharmacokinetics of several FZD8-Fc variants were assessed in ratsin a two week pharmacokinetics (PK) study. The FZD8-Fc variantsevaluated were 54F03, 54F09, 54F12, 54F13, 54F15 and 54F16. SpragueDawley rats, five males in each group, were dosed with FZD8-Fc variantsvia the tail vein at 10 mg/kg and followed for two weeks with samplescollected at time points 1, 24, 48, 72, 96, 168, 240, and 336 hours. Ateach time point, 1 ml of blood was collected into potassium-EDTA tubesand centrifuged. The plasma supernatants were collected and frozen untilthe samples were analyzed.

The level of FZD8-Fc variant protein present in the plasma at each timepoint was quantified and the half-life of each FZD8-Fc variant wascalculated. The half-life of the FZD8-Fc variants is shown in Table 4.

TABLE 4 FZD8-Fc Variant t_(1/2) in hours Fc Region 54F03 162 IgG1 54F09136 IgG1 54F12 152 IgG2 54F13 268 IgG2 54F15 109 IgG1 54F16 154 IgG1

Example 13 Pharmacokinetics of FZD8-Fc Variants in Cynomolgus Monkeys

Four young adult/adult male naïve cynomolgus monkeys were randomlydivided into two groups of two, and administered an intravenous (IV)bolus of FZD-Fc variant 54F 15 or variant 54F 16 at a dose of 30 mg/kg.Twice daily (a.m. and p.m.) animals were observed for mortality andsigns of pain and distress. Cageside observations for general health andappearance were done once daily. On the day of dosing, each animal wasobserved at approximately 1 and 4 hours post-dose for mortality andsigns of pain and distress. Any unusual observations noted throughoutthe duration of the study were recorded. Body weights were taken on theday of dose administration and at the end of blood collection. Blood(approximately 0.5 ml) was collected from a femoral vein via syringe andneedle and transferred into tubes containing EDTA K3 anticoagulantpre-dose and at 1, 6, 12, 24, 48, 72, 96, 168, 240, and 336 hourspost-dose for PK analysis. In addition, blood (approximately 0.5 ml) wascollected from a femoral vein via syringe and needle and transferredinto tubes containing no anticoagulant pre-dose and at 336 hourspost-dose for anti-drug antibody (ADA) analysis. Plasma supernatantswere collected and frozen until the samples were analyzed. HTRF(homogeneous time resolved fluorescence) immunoassays were performed todetermine the FZD-Fc concentration in animal plasma samples for PKanalysis and the concentration of anti-drug antibody in serum. FZD-Fcconcentration in plasma versus time was analyzed by non-compartmentalanalysis (NCA) with Phoenix™ WinNonlin® Version 6.0, using a bolus IVadministration model.

The level of FZD8-Fc variants present in the plasma at each time pointwas quantified (FIG. 16) and the half-life of FZD8-Fc variants 54F15 and54F16 was calculated. As shown in Table 5, the half-life of FZD8-Fcvariant 54F15 was estimated to be 102 hours at 30 mg/kg and thehalf-life of FZD-Fc8 variant 54F16 was estimated to be 137 hours at 30mg/kg.

TABLE 5 Animal T_(1/2λz) AUC_(0-last) AUC_(0-∞) AUC % Vz Cl ID (hr)(ng*hr/ml) (ng*hr/ml) Extrap (ml) (ml/hr) FZD8-Fc 54F15 C43064 102.328642771.4 31231858.7 8.3 141.7 0.960 C43061 102.5 31904918.2 34846234.18.4 127.3 0.861 Mean 102.4 30273845 33039046 8.35 134.5 0.9105 FZD8-Fc54F16 C43066 139.0 29088083.0 35470395.6 18.0 169.6 0.846 C43076 134.135934159.0 43449944.6 17.3 133.6 0.690 Mean 136.6 32511121 3946017017.65 151.6 0.768

Example 14 Inhibition of Colon Tumor Growth in vivo by FZD8-Fc Variants

Dissociated C28 colon tumor cells (10,000 cells) were injectedsubcutaneously into 6-8 week old male NOD/SCID mice. Tumors were allowedto grow for 28 days until they reached an average volume of 145 mm³. Themice were randomized (n=9 per group) and treated with FZD8-Fc variantsor a control antibody at a dose of 15mg/kg twice a week. Administrationof the FZD8-Fc variants and control antibody was performed via injectioninto the intraperitoneal cavity. Tumor growth was monitored and tumorvolumes were measured with electronic calipers at the indicated timepoints. Data are expressed as mean±S.E.M.

Variants 54F12 and 54F13 had no apparent effect upon tumor growth, withtumor volumes substantially the same as the tumors in mice treated withcontrol antibody. In contrast, treatment with variants 54F03, 54F09,54F15 and 54F16 resulted in approximately 56%, 70%, 64% and 70%reduction (respectively) in tumor growth as compared to treatment withthe control antibody, as shown in FIG. 17. Thus, the anti-tumor growthactivity of the FZD8-Fc variants appeared to be affected by the aminoacid sequence at the junction between the FZD8 portion and the Fcportion. In addition, anti-tumor growth activity appeared to be affectedby the source of the Fc region, as both variants that are IgG2 fusionproteins (54F12 and 54F13), did not inhibit tumor growth in this model.

Example 15 Inhibition of Pancreatic Tumor Growth in vivo by FZD8-FcVariants

Dissociated PN4 pancreatic tumor cells (10,000 cells) were injectedsubcutaneously into 6-8 week old male NOD/SCID mice. Tumors were allowedto grow for 36 days until they reached an average volume of 112 mm³. Themice were randomized (n=10 per group) and treated with FZD8-Fc variantsor a control antibody at a dose of 15 mg/kg twice a week. Administrationof the FZD8-Fc variants and control antibody was performed via injectioninto the intraperitoneal cavity. Tumor growth was monitored and tumorvolumes were measured with electronic calipers at the indicated timepoints. Data are expressed as mean±S.E.M.

Variants 54F12 and 54F13 reduced tumor growth less than 20% as comparedwith tumors in mice treated with control antibody. Treatment withFZD8-Fc variants 54F03, 54F09, 54F15 and 54F16 reduced tumor growthapproximately 20% to 60% as compared to treatment with the controlantibody. As shown in FIG. 18, variants 54F09 and 54F16 reduced tumorgrowth by the greatest amount, 45% (p <0.001) and 60% (p<0.001),respectively. Thus, the anti-tumor growth activity of the FZD8-Fcvariants appeared to be affected by the amino acid sequence at thejunction between the FZD8 portion and the Fc portion. As seen in theExample 14, anti-tumor growth activity also appeared to be affected bythe source of the Fc region, as both variants that are IgG2 fusionproteins (54F12 and 54F13) had weaker anti-tumor activity than theFZD8-Fc variants that are IgG1 fusion proteins.

Pancreatic tumor cells from the tumor-bearing mice described above wereharvested and minced into approximately 1 mm³ fragments, followed byenzymatic digestion at 1 gram per 10 ml of 300 μg/ml collagenase and 200U/ml DNase I for 2 hours at 37° C./5% CO₂ with intermittent mixing witha 10m1 pipet to disperse cells. Digestion was stopped by adding an equalvolume of FACS buffer (1× Hanks Buffered Saline Solution (HBSS), 2%heat-inactivated Fetal Calf Serum (FCS) and 2 mM HEPES pH 7.4). Cellswere filtered through 40 μm nylon filters and collected bycentrifugation at 150×g for 5 minutes. Red blood cells were lysed in ahypotonic buffer containing ammonium chloride for 2 minutes on ice, andthe cells were washed again with excess FACS buffer, and resuspendedwith FACS buffer at 1×10⁷ cells/ml.

The freshly prepared single cell suspensions were stained for 20 minuteson ice with biotinylated anti-mouse H-2Kd (clone SF1-1.1, Biolegend, SanDiego, Calif.) at 5 μg/ml, biotinylated anti-mouse CD45 (30-F11,Biolegend) at 2.5 μm/ml, and streptavidin-PerCP-Cy5.5 (eBioscience, SanDiego, Calif.) at 1:200 dilution. Unbound antibody was removed bywashing twice with 10 volume of FACS buffer. For analysis of human cellsurface markers, the single tumor cell suspension was stained withanti-ESA-FITC (Miltenyi Biotec, Auburn, Calif.) at 1:50 dilution,anti-human CD44-PE-Cy7 (eBioscience, San Diego, Calif.) at 1:100dilution, and anti-human CD201-PE (BD Biosciences) at 1:5 dilution. Thecells are washed, and resuspended in FACS buffer containing 2.5 ug/ml of4′-6-Diamidino-2-phenylindole (DAPI). Cells stained with a singlefluorescent color were used for instrument calibration. Any remainingmouse cells (positive for H-2Kd and CD45) and dead cells (DAPI-positive)were excluded during cell sorting. Cell doublets and clumps wereexcluded using doublet discrimination gating.

As shown in FIG. 19, treatment of tumor-bearing mice with FZD8-Fcvariants 54F03 and 54F16 decreased the percentage of CD44^(hi) cells ascompared to mice treated with the control antibody. Although thepercentage of CD201⁺CD44⁺ cells was small, treatment of tumor-bearingmice with FZD8-Fc variants 54F03 and 54F16 decreased the percentage ofCD201⁺CD44⁺ cells as compared to mice treated with the control antibody.CD44 has been shown to be to marker of tumorigenic cells (e.g. cancerstem cells). In addition, in some embodiments, cells that areCD44^(hi)CD201⁺ have been found to be more tumorigenic thanCD44^(hi)CD201⁻ cells. Thus, it is important that FZD8-Fc variants werecapable of decreasing the percentage of both CD44^(hi) andCD44^(hi)CD201⁺ cell populations, thereby decreasing the percentage ornumber of tumorigenic cells in the treated mice.

Example 16 Characterization of N-Termini

The correct signal sequence cleavage site in the FZD8 protein ispredicted to be between amino acid 25 (an alanine) and amino acid 26 (analanine); cleavage at this site leaves an N-termini of ASA. Analysis bymass spectrometry was used to determine the mass of FZD8-Fc proteins ascompared to the theoretical mass of the FZD8-Fc protein cleaved at thepredicted site.

Additional FZD8-Fc variants with modified signal sequences were producedat DNA2.0 (Menlo Park, Calif.) as described above. DNA2.0 synthesizedand assembled short single-stranded oligonucleotides to produce theFZD8-Fc variant proteins, 54F23 to 54F35. The assembled oligonucleotideswere subsequently cloned and sequence verified.

Plasmid DNA of each FZD8-Fc variant was prepared using QIAGEN maxi-prepkits following the manufacturer's protocol. Expression of each variantwas done using FreeStyle™ MAX reagent (Life Technologies) and 293FScells. Cells were grown to log phase and diluted to 1×10⁶ cell/ml. Foreach reaction, 315 ug of plasmid DNA was diluted into 5 ml of OptiMEMPro. In a different tube, 315 ul of FreeStyle™ MAX reagent was dilutedinto 5 ml of OptiMem Pro. Plasmid DNA was complexed with the FreeStyle™MAX reagent by adding the diluted reagent dropwise to the DNA, followedby an incubation of 15 minutes at room temperature. The DNA-reagentcomplex was then added to 250 ml of 293FS cells. Expression reactionswere allowed to grow for 7-10 days, at which time, they were harvestedby centrifugation and filtration. Each Fzd8-Fc variant was purified byaffinity purification using a 5 ml HiTrap MAbSelect SURE column.Briefly, harvested media were passed through columns that had beenequilibrated with binding buffer. The columns were washed with bindingbuffer to remove unbound material, and then FZD8-Fc protein was elutedwith elution buffer. Eluted samples were then dialyzed into a buffersuitable for mass spectrometry.

Approximately 250 μg of each FZD8-Fc sample was reduced with Tris(2-carboxyethyl) phosphine (TCEP) for 30 minutes at 37° C. to separateheavy and light chains of the antibody. Reduced samples were thenalkylated by treatment with iodoacetamide for 30 minutes at 37° C.Samples were passed over NAP-5 columns (GE Health Care) to change thebuffer to 10 mM Tris-HCL (pH 7.4). Buffer exchange was followeddeglycosylation with endoglycosidase PNGase F. Samples were incubatedovernight at 37° C. at a ratio of 1:200 (enzyme:sample). The reactionswere stopped by addition of acid. The reduced, alkylated, anddeglycosylated FZD8-Fc samples were loaded into vials for liquidchromatography-mass spectrometry (LC/MS) analysis using a Waters UPLC™and electrospray-QToF mass spectrometer. Mass calibration of each sampleanalysis using cesium trifluoroacetic acid ion clusters was conductedwith a Waters LockSpray™ dual electrospray ion source.

As shown in FIG. 20A, a FZD8-Fc protein (54F16) with a signal sequencethat was the same sequence as the native sequence was produced as aheterogeneous mixture in regard to the N-terminal sequence. Aproportional of the protein present in sample was equivalent in mass toa protein cleaved at amino acids 25 and 26 (peak at 41704.0) with anN-terminal sequence of ASA. However, greater than 50% of the protein waspresent in a form with a different mass (peak at 41918.2). This peakmost likely represents a protein cleaved at amino acids 22 and 23;cleavage at this site leaves an N-terminal sequence of AAAASA (SEQ IDNO:76).

FZD8-Fc variants with signal sequences SEQ ID NO:68 to SEQ ID NO:74 weregenerated, purified from cell culture, and analyzed by mass spectrometryas described above. It was observed that variants with signal sequencesSEQ ID NO:70 to SEQ ID NO:74 produced an almost completely homogeneousprotein sample (several representative results are shown in FIGS.20B-20E). FZD8-Fc variant 54F26 which comprises SEQ ID NO:53 with signalsequence SEQ ID NO:71 was present predominantly (greater than 95%) as aprotein cleaved at amino acids 25 and 26 (peak 41930.1) with anN-terminal sequence of ASA (FIG. 20B). Similar results were also seenwith a variant comprising SEQ ID NO:53 and signal sequence SEQ ID NO:72(54F28, FIG. 20C), a variant comprising SEQ ID NO:53 and signal sequenceSEQ ID NO:73 (54F30, FIG. 20D) and a variant comprising SEQ ID NO:53 andsignal sequence SEQ ID NO:74 (54F32, FIG. 20E). Similar results wereobserved with proteins produced from transient and stable transfections.

Example 17 Inhibition of Colon Tumor Growth in vivo by FZD8-Fc Variants

Dissociated C28 colon tumor cells (10,000 cells) were injectedsubcutaneously into 6-8 week old male NOD/SCID mice. Tumors were allowedto grow for 56 days until they reached an average volume of 175 mm³. Themice were randomized (n=10 per group) and treated with FZD8-Fcconstructs 54F03, 54F23, 54F26, or a control antibody at a dose of 15mg/kg twice a week. Administration of the FZD8-Fc variants and controlantibody was performed via injection into the intraperitoneal cavity.Tumor growth was monitored and tumor volumes were measured withelectronic calipers at the indicated time points. Data are expressed asmean±S.E.M.

FZD8-Fc variants 54F23 and 54F26 are produced as predominantly ahomogenous protein with an N-terminus of amino acids ASA, while 54F03 isproduced as a heterogeneous protein mixture with N-termini of aminoacids ASA and AAAASA. As shown in FIG. 21 treatment with FZD8-Fcvariants 54F03, 54F23, and 54F26 reduced tumor growth 48%, 57% and 52%,respectively, as compared to treatment with the control antibody afterthree weeks of treatment.

All publications, patents, patent applications, internet sites, andaccession numbers/database sequences (including both polynucleotide andpolypeptide sequences) cited herein are hereby incorporated by referencein their entirety for all purposes to the same extent as if eachindividual publication, patent, patent application, internet site, oraccession number/database sequence were specifically and individuallyindicated to be so incorporated by reference.

SEQUENCES FZD8-Fc amino acid sequence-variant 54F03(without predicted signal sequence; the “GRA”linker sequence between the FZD8 sequence andthe Fc sequence of the fusion protein is underlined) (SEQ ID NO: 1)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KFZD8-Fc coding sequence (nucleotides encoding FZD8-derived sequences are underlined) (SEQ ID NO: 2)ATGGAGTGGGGTTACCTGTTGGAAGTGACCTCGCTGCTGGCCGCCTTGGCGCTGCTGCAGCGCTCTAGCGGCGCTGCGGCCGCCTCGGCCAAGGAGCTGGCATGCCAAGAGATCACCGTGCCGCTGTGTAAGGGCATCGGCTACAACTACACCTACATGCCCAATCAGTTCAACCACGACACGCAAGACGAGGCGGGCCTGGAGGTGCACCAGTTCTGGCCGCTGGTGGAGATCCAGTGCTCGCCCGATCTCAAGTTCTTCCTGTGCAGCATGTACACGCCCATCTGCCTAGAGGACTACAAGAAGCCGCTGCCGCCCTGCCGCTCGGTGTGCGAGCGCGCCAAGGCCGGCTGCGCGCCGCTCATGCGCCAGTACGGCTTCGCCTGGCCCGACCGCATGCGCTGCGACCGGCTGCCCGAGCAAGGCAACCCTGACACGCTGTGCATGGACTACAACCGCACCGACCTAACCACCGGGCGCGCCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Minimum FZD and SFRP Fri domain sequencesh-FZD1 amino acids 116-227 (SEQ ID NO: 3)CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCh-FZD2 amino acids 39-150 (SEQ ID NO: 4)CQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICh-FZD3 amino acids 28-133 (SEQ ID NO: 5)CEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDC h-FZD4 amino acids 48-161(SEQ ID NO: 6)CDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCh-FZD5 amino acids 33-147 (SEQ ID NO: 7)CQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCh-FZD6 amino acids 24-129 (SEQ ID NO: 8)CEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYC h-FZD7 amino acids 49-160(SEQ ID NO: 9)CQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICh-FZD8 amino acids 35-148 (SEQ ID NO: 10)CQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCh-FZD9 amino acids 39-152 (SEQ ID NO: 11)CQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCh-FZD10 amino acids 34-147 (SEQ ID NO: 12)CQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCh-SFRP1 amino acids 57-165 (SEQ ID NO: 13)CVDIPADLRLCHNVGYKKMVLPNLLEHETMAEVKQQASSWVPLLNKNCHAGTQVFLCSLFAPVCLDRPIYPCRWLCEAVRDSCEPVMQFFGFYWPEMLKCDKFPEGDVCh-SFRP2 amino acids 40-152 (SEQ ID NO: 14)CKPIPANLQLCHGIEYQNMRLPNLLGHETMKEVLEQAGAWIPLVMKQCHPDTKKFLCSLFAPVCLDDLDETIQPCHSLCVQVKDRCAPVMSAFGFPWPDMLECDRFPQDNDLCh-SFRP3 amino acids 35-147 (SEQ ID NO: 15)CEPVRIPLCKSLPWNMTKMPNHLHHSTQANAILAIEQFEGLLGTHCSPDLLFFLCAMYAPICTIDFQHEPIKPCKSVCERARQGCEPILIKYRHSWPENLACEELPVYDRGVCh-SFRP4 amino acids 24-136 (SEQ ID NO: 16)CEAVRIPMCRHMPWNITRMPNHLHHSTQENAILAIEQYEELVDVNCSAVLRFFFCAMYAPICTLEFLHDPIKPCKSVCQRARDDCEPLMKMYNHSWPESLACDELPVYDRGVCh-SFRP5 amino acids 53-162 (SEQ ID NO: 17)CLDIPADLPLCHTVGYKRMRLPNLLEHESLAEVKQQASSWLPLLAKRCHSDTQVFLCSLFAPVCLDRPIYPCRSLCEAVRAGCAPLMEAYGFPWPEMLHCHKFPLDNDLC Fc sequencesHuman IgG₁ Fc region (SEQ ID NO: 18)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region(SEQ ID NO: 42)KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG₁ Fc region(SEQ ID NO: 43)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman IgG₂ Fc region (SEQ ID NO: 44)CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FZD Fri domain sequencesHuman FZD4 Fri domain (predicted signal sequence underlined)(SEQ ID NO: 19)MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVHuman FZD5 Fri domain (predicted signal sequence underlined)(SEQ ID NO: 20)MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTHuman FZD8 Fri domain (predicted signal sequence underlined)(SEQ ID NO: 21)MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTHuman FZD1 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 32 amino acids 87-237 ofSEQ ID NO: 27)QQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTHuman FZD2 Fri domain amino acid sequencewithout predicted signal sequence; SEQ ID NO: 33 amino acids 24-159 ofSEQ ID NO: 28)QFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDG Human FZD3 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 34 amino acids 23-143 ofSEQ ID NO: 29) HSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPY PRLVDLHuman FZD4 Fri domain amino acid sequencewithout predicted signal sequence e; (SEQ ID NO: 35amino acids 40-170 of SEQ ID NO: 22)FGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNH MCMEGPGDEEVHuman FZD5 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 36 amino acids 27-157 ofSEQ ID NO: 23)ASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVL CMDYNRSEATTHuman FZD6 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 37 amino acids 19-146 ofSEQ ID NO: 24)HSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFD PHTEFLGHuman FZD7 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 38 amino acids 33-170 ofSEQ ID NO: 25)QPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSG Human FZD8 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 39 amino acids 28-158 ofSEQ ID NO: 30)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTL CMDYNRTDLTTHuman FZD9 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 40 amino acids 23-159 ofSEQ ID NO: 31)LEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENA Human FZD10 Fri domain amino acid sequencewithout predicted signal sequence; (SEQ ID NO: 41 amino acids 21-154 ofSEQ ID NO: 26)ISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNG FZD extracellular domain (ECD) sequencesHuman FZD1 ECD with signal sequence (SEQ ID NO: 27)MAEEEAPKKSRAAGGGASWELCAGALSARLAEEGSGDAGGRRRPPVDPRRLARQLLLLLWLLEAPLLLGVRAQAAGQGPGQGPGPGQQPPPPPQQQQSGQQYNGERGISVPDHGYCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSAELKFFLCSMYAPVCTVLEQALPPCRSLCERARQGCEALMNKFGFQWPDTLKCEKFPVHGAGELCVGQNTSDKGTPTPSLLPEFWTSNPQHGGGGHRGGFPGGAGASERGKFSCPRALKVPSYLNYHFLGEKDCGAPCEPTKVYGLMYFGPEELRFSRT Human FZD2 ECD with signal sequence(SEQ ID NO: 28)MRPRSALPRLLLPLLLLPAAGPAQFHGEKGISIPDHGFCQPISIPLCTDIAYNQTIMPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLEQAIPPCRSICERARQGCEALMNKFGFQWPERLRCEHFPRHGAEQICVGQNHSEDGAPALLTTAPPPGLQPGAGGTPGGPGGGGAPPRYATLEHPFHCPRVLKVPSYLSYKFLGERDCAAPCEPARPDGSMFFSQEE TRFARLWILTHuman FZD3 ECD with signal sequence (SEQ ID NO: 29)MAMTWIVFSLWPLTVFMGHIGGHSLFSCEPITLRMCQDLPYNTTFMPNLLNHYDQQTAALAMEPFHPMVNLDCSRDFRPFLCALYAPICMEYGRVTLPCRRLCQRAYSECSKLMEMFGVPWPEDMECSRFPDCDEPYPRLVDLNLAGEPTEGAPVAVQRDYGFWCPRELKIDPDLGYSFLHVRDCSPPCPNMYFRREELSFARY Human FZD4 ECD with signal sequence(SEQ ID NO: 22)MLAMAWRGAGPSVPGAPGGVGLSLGLLLQLLLLLGPARGFGDEEERRCDPIRISMCQNLGYNVTKMPNLVGHELQTDAELQLTTFTPLIQYGCSSQLQFFLCSVYVPMCTEKINIPIGPCGGMCLSVKRRCEPVLKEFGFAWPESLNCSKFPPQNDHNHMCMEGPGDEEVPLPHKTPIQPGEECHSVGTNSDQYIWVKRSLNCVLKCGYDAGLYSRSAKEFTDIHuman FZD5 ECD with signal sequence (SEQ ID NO: 23)MARPDPSAPPSLLLLLLAQLVGRAAAASKAPVCQEITVPMCRGIGYNLTHMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLRFFLCSMYTPICLPDYHKPLPPCRSVCERAKAGCSPLMRQYGFAWPERMSCDRLPVLGRDAEVLCMDYNRSEATTAPPRPFPAKPTLPGPPGAPASGGECPAGGPFVCKCREPFVPILKESHPLYNKVRTGQVPNCAVPCYQPSFSADERTHuman FZD6 ECD with signal sequence (SEQ ID NO: 24)MEMFTFLLTCIFLPLLRGHSLFTCEPITVPRCMKMAYNMTFFPNLMGHYDQSIAAVEMEHFLPLANLECSPNIETFLCKAFVPTCIEQIHVVPPCRKLCEKVYSDCKKLIDTFGIRWPEELECDRLQYCDETVPVTFDPHTEFLGPQKKTEQVQRDIGFWCPRHLKTSGGQGYKFLGIDQCAPPCPNMYFKSDELEFAKSFIGTVSI Human FZD7 ECD with signal sequence(SEQ ID NO: 25)MRDPGAAAPLSSLGLCALVLALLGALSAGAGAQPYHGEKGISVPDHGFCQPISIPLCTDIAYNQTILPNLLGHTNQEDAGLEVHQFYPLVKVQCSPELRFFLCSMYAPVCTVLDQAIPPCRSLCERARQGCEALMNKFGFQWPERLRCENFPVHGAGEICVGQNTSDGSGGPGGGPTAYPTAPYLPDLPFTALPPGASDGRGRPAFPFSCPRQLKVPPYLGYRFLGERDCGAPCEPGRANGLMYFKEEERRFARL Human FZD8 ECD with signal sequence (SEQ ID NO: 30)MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPRRLPPPPPGEQPPSGSGHGRPPGARPPHRGGGRGGGGGDAAAPPARGGGGGGKARPPGGGAAPCEPGCQCRAPMVSVSSERHPLYNRVKTGQIANCALPCHNPFFSQDERAFTHuman FZD9 ECD with signal sequence (SEQ ID NO: 31)MAVAPLRGALLLWQLLAAGGAALEIGRFDPERGRGAAPCQAVEIPMCRGIGYNLTRMPNLLGHTSQGEAAAELAEFAPLVQYGCHSHLRFFLCSLYAPMCTDQVSTPIPACRPMCEQARLRCAPIMEQFNFGWPDSLDCARLPTRNDPHALCMEAPENATAGPAEPHKGLGMLPVAPRPARPPGDLGPGAGGSGTCENPEKFQYVEKSRSCAPRCGPGVEVFWSRRDKDFHuman FZD10 ECD with signal sequence (SEQ ID NO: 26)MQRPGPRLWLVLQVMGSCAAISSMDMERPGDGKCQPIEIPMCKDIGYNMTRMPNLMGHENQREAAIQLHEFAPLVEYGCHGHLRFFLCSLYAPMCTEQVSTPIPACRVMCEQARLKCSPIMEQFNFKWPDSLDCRKLPNKNDPNYLCMEAPNNGSDEPTRGSGLFPPLFRPQRPHSAQEHPLKDGGPGRGGCDNPGKFHHVEKSASCAPLCTPGVDVYWSREDKRFA FZD8-Fc variantsFZD8-Fc variant 54F03 amino acid sequence(without predicted signal sequence; alternative cleavage)(SEQ ID NO: 45)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTGRADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKFZD8-Fc variant 54F09 amino acid sequence(without predicted signal sequence) (SEQ ID NO: 46)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKFZD8-Fc variant 54F09 amino acid sequence(without predicted signal sequence; alternative cleavage)(SEQ ID NO: 47)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPSPPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGKFZD8-Fc variant 54F15 amino acid sequence(without predicted signal sequence) (SEQ ID NO: 48)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KFZD8-Fc variant 54F15 amino acid sequence(without predicted signal sequence; alternative cleavage)(SEQ ID NO: 49)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTAAPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGKFZD8-Fc variant 54F16, 54F17, 54F18, 54F23,54F25, 54F27, 54F29, 54F31, and 54F34amino acid sequence (without predicted signal sequence) (SEQ ID NO: 50)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KFZD8-Fc variant 54F16 amino acid sequence(without predicted signal sequence; alternative cleavage)(SEQ ID NO: 51)AAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGFZD8-Fc variant 54F16 amino acid sequence (with signal sequence)(SEQ ID NO: 52)MEWGYLLEVTSLLAALALLQRSSGAAAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK FZD8-Fc variant 54F19, 54F20, 54F24, 54F26,54F28, 54F30, 54F32, 54F34 and 54F35amino acid sequence (without predicted signal sequence) (SEQ ID NO: 53)ASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKFZD8-Fc variant 54F19 amino acid sequence(without predicted signal sequence; alternative cleavage)(SEQ ID NO: 54)ALAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKFZD8-Fc variant 54F20 amino acid sequence(without predicted signal sequence; alternative cleavage)(SEQ ID NO: 55)VLAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKFZD8-Fc variant 54F34 amino acid sequence(without predicted signal sequence) (SEQ ID NO: 65)KELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFZD8-Fc variant 54F33 aminoa cid sequence(without predicted signal sequence) (SEQ ID NO: 66)KELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHuman ROR1 ECD with signal sequence (SEQ ID NO: 56)MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYHuman ROR2 ECD with signal sequence (SEQ ID NO: 57)MARGSALPRRPLLCIPAVWAAAALLLSVSRTSGEVEVLDPNDPLGPLDGQDGPIPTLKGYFLNFLEPVNNITIVQGQTAILHCKVAGNPPPNVRWLKNDAPVVQEPRRIIIRKTEYGSRLRIQDLDTTDTGYYQCVATNGMKTITATGVLFVRLGPTHSPNHNFQDDYHEDGFCQPYRGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSRTPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPESPDAANCMRIGIPAERLGRYHQCYNGSGMDYRGTASTTKSGHQCQPWALQHPHSHHLSSTDFPELGGGHAYCRNPGGQMEGPWCFTQNKNVRMELCDVPSCSPRDSSKMG h-ROR1 minimal Fri domain(SEQ ID NO: 58)CQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQPE SPEAANCh-ROR2 minimal Fri domain (SEQ ID NO: 59)CQPYRGIACARFIGNRTIYVDSLQMQGEIENRITAAFTMIGTSTHLSDQCSQFAIPSFCHFVFPLCDARSRTPKPRELCRDECEVLESDLCRQEYTIARSNPLILMRLQLPKCEALPMPE SPDAANCLinker (SEQ ID NO: 60) ESGGGGVT Linker (SEQ ID NO: 61) LESGGGGVT Linker(SEQ ID NO: 62) GRAQVT Linker (SEQ ID NO: 63) WRAQVT Linker(SEQ ID NO: 64) ARGRAQVT Signal Sequence (SEQ ID NO: 67)MEWGYLLEVTSLLAALALLQRSSGAAA Signal Sequence (SEQ ID NO: 68)MEWGYLLEVTSLLAALALLQRSSGALA Signal Sequence (SEQ ID NO: 69)MEWGYLLEVTSLLAALALLQRSSGVLA Signal Sequence (SEQ ID NO: 70)MEWGYLLEVTSLLAALLLLQRSPIVHA Signal Sequence (SEQ ID NO: 71)MEWGYLLEVTSLLAALFLLQRSPIVHA Signal Sequence (SEQ ID NO: 72)MEWGYLLEVTSLLAALLLLQRSPFVHA Signal Sequence (SEQ ID NO: 73)MEWGYLLEVTSLLAALLLLQRSPIIYA Signal Sequence (SEQ ID NO: 74)MEWGYLLEVTSLLAALLLLQRSPIAHA FZD8-Fc variant 54F26 with signal sequence(SEQ ID NO: 75)MEWGYLLEVTSLLAALFLLQRSPIVHAASAKELACQEITVPLCKGIGYNYTYMPNQFNHDTQDEAGLEVHQFWPLVEIQCSPDLKFFLCSMYTPICLEDYKKPLPPCRSVCERAKAGCAPLMRQYGFAWPDRMRCDRLPEQGNPDTLCMDYNRTDLTTEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N-terminal sequence (SEQ ID NO: 76)AAAASA

1-184. (canceled)
 185. A Wnt-binding agent comprising: (a) a firstpolypeptide consisting essentially of SEQ ID NO:32; SEQ ID NO:33; SEQ IDNO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ IDNO:39; SEQ ID NO:40; or SEQ ID NO:41; and (b) a second polypeptideconsisting essentially of SEQ ID NO:42 or SEQ ID NO:43; wherein thefirst polypeptide is directly linked to the second polypeptide.
 186. TheWnt-binding agent of claim 185, wherein the first polypeptide consistsessentially of SEQ ID NO:39.
 187. The Wnt-binding agent of claim 185,wherein the first polypeptide consists of SEQ ID NO:39; and wherein thesecond polypeptide consists of SEQ ID NO:42 or SEQ ID NO:43.
 188. Apolypeptide comprising the Wnt-binding agent of claim 186, and furthercomprising an amino acid sequence selected from the group consisting of:SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71,SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74.
 189. A cell producing theWnt-binding agent of claim
 185. 190. A composition comprising theWnt-binding agent of claim 186, wherein at least about 80% of theWnt-binding agent has an N-terminal amino acid sequence of ASA.
 191. Apharmaceutical composition comprising the Wnt-binding agent of claim 185and a pharmaceutically acceptable carrier.
 192. A polynucleotidecomprising a polynucleotide that encodes the Wnt-binding agent of claim185.
 193. A polynucleotide comprising a polynucleotide that encodes thepolypeptide of claim
 188. 194. A method of inhibiting the growth of atumor in a subject, comprising administering to the subject atherapeutically effective amount of the Wnt-binding agent of claim 185.195. A method of treating cancer in a subject, comprising administeringto the subject a therapeutically effective amount of the Wnt-bindingagent of claim
 185. 196. A method of treating a disease in a subjectwherein the disease is associated with Wnt signaling activation,comprising administering to the subject a therapeutically effectiveamount of the Wnt-binding agent of claim
 185. 197. A method of inducingdifferentiation of tumor cells, comprising contacting the tumor cellswith an effective amount of the Wnt-binding agent of claim
 185. 198. Amethod of reducing the frequency of cancer stem cells in a tumor,comprising contacting the tumor cells with an effective amount of theWnt-binding agent of claim
 185. 199. A method of producing theWnt-binding agent of claim 185, wherein at least about 80% of theWnt-binding agent has an N-terminal amino acid sequence of ASA, themethod comprising using a signal sequence selected from the groupconsisting of: SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70,SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, and SEQ ID NO:74 forproduction of the Wnt-binding agent.
 200. A Wnt-binding agentcomprising: (a) a first polypeptide consisting essentially of aminoacids X to Y of SEQ ID NO:30; and (b) a second polypeptide consistingessentially of amino acids A to B of SEQ ID NO:43; wherein the firstpolypeptide is directly linked to the second polypeptide; and whereinX=amino acid 25, 26, 27, 28, 29, 30, or 31 Y=amino acid 156, 157, 158,159, 160, 161, 162, 163, or 164 A=amino acid 1, 2, 3, 4, 5, or 6 B=aminoacid 231 or
 232. 201. A polypeptide comprising an amino acid sequenceselected from the group consisting of: SEQ ID NO:46, SEQ ID NO:48, SEQID NO:50, and SEQ ID NO:53.
 202. The polypeptide of claim 201, whereinthe amino acid sequence is SEQ ID NO:50 or SEQ ID NO:53.
 203. Thepolypeptide of claim 201, which further comprises an amino acid sequenceselected from the group consisting of: SEQ ID NO:67, SEQ ID NO:68, SEQID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, andSEQ ID NO:74.
 204. A composition comprising the polypeptide of claim201, wherein at least 80% of the polypeptide has an N-terminal aminoacid sequence of ASA.
 205. An isolated polynucleotide comprising apolynucleotide that encodes the polypeptide of claim
 201. 206. Anisolated polynucleotide comprising a polynucleotide that encodes thepolypeptide of claim
 203. 207. An isolated polynucleotide comprising apolynucleotide that encodes a polypeptide comprising: (a) a signalsequence selected from the group consisting of SEQ ID NO:67, SEQ IDNO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, and SEQ ID NO:74; (b) a Fri domain of human FZD8; and (c) a humanFc region.
 208. A cell comprising the polynucleotide of claim
 207. 209.A polypeptide produced by the cell of claim
 208. 210. The polypeptide ofclaim 209, wherein at least about 80% of the polypeptide has anN-terminal amino acid sequence of ASA.
 211. A method of producing asoluble Wnt-binding agent which comprises a Fri domain of human FZD8 ina cell, wherein at least about 80% of the Wnt-binding agent has anN-terminal amino acid sequence of ASA, the method comprising using asignal sequence selected from the group consisting of: SEQ ID NO:67, SEQID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ IDNO:73, and SEQ ID NO:74 for production of the Wnt-binding agent.
 212. Asoluble Wnt-binding agent produced by the method of claim
 211. 213. Acomposition comprising a soluble Wnt-binding agent which comprises a Fridomain of human FZD8, wherein at least about 80% of the Wnt-bindingagent has an N-terminal sequence of ASA.
 214. A method of treatingcancer in a subject, comprising administering to the subject atherapeutically effective amount of the composition of claim
 213. 215. Amethod of treating a disease in a subject wherein the disease isassociated with Wnt signaling activation, comprising administering tothe subject a therapeutically effective amount of the composition ofclaim
 213. 216. A method of screening an agent that binds one or moreWnt proteins for anti-tumor activity, comprising: (a) exposing a firstsolid tumor, but not a second solid tumor, to the agent; (b) assessingthe levels of one or more differentiation markers and/or one or moresternness markers in the first and second solid tumors; and (c)comparing the levels of the one or more differentiation markers and/orone or more sternness markers in the first tumor to the levels of theone or more differentiation markers and/or one or more sternness markersin the second solid tumor.
 217. The method of claim 216, wherein (i)increased levels of the one or more differentiation markers in the firstsolid tumor relative to the levels of the one or more differentiationmarkers in the second solid tumor indicate anti-tumor activity of theagent; and (ii) decreased levels of the one or more sternness markersindicate anti-tumor activity of the agent.
 218. A polypeptide comprisingan amino acid sequence selected from the group consisting of: SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, and SEQ ID NO:74.
 219. A polynucleotide comprisinga polynucleotide encoding the polypeptide of claim 218.